Transgenic plants having increased tolerance to aluminum

ABSTRACT

Methods and materials for modulating aluminum tolerance in plants are disclosed. For example, nucleic acids encoding aluminum tolerance-modulating polypeptides are disclosed as well as methods for using such nucleic acids to transform plant cells. Also disclosed are plants having increased tolerance to aluminum and methods of increasing plant yield in soil containing elevated levels of aluminum.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a National Stage Application under 35U.S.C. § 371and claims the benefit of International Application No.PCT/US2012/062977, filed Nov. 1, 2012, which claims priority to U.S.Application Ser. No. 61/554,788, filed on Nov. 2, 2011, entitledTRANSGENIC PLANTS HAVING INCREASED TOLERANCE TO ALUMINUM, thedisclosures of which are incorporated herein by reference.

TECHNICAL FIELD

This document relates to methods and materials involved in modulatingtolerance to abiotic stress in plants. For example, this documentprovides plants having increased aluminum tolerance, materials andmethods for making plants having increased aluminum tolerance, as wellas methods of increasing plant yield in soil containing elevated levelsof aluminum.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The material in the accompanying sequence listing is hereby incorporatedby reference into this application. The accompanying file, namedSequence_Listing_2012-10-30.txt was created on Oct. 30, 2012, and is1.69 MB. The file can be accessed using Microsoft Word on a computerthat uses Windows OS.

BACKGROUND

Aluminum (Al) is ubiquitous in soils and at pH values below 5.0, issolubilized into the soil solution as the highly phytotoxic Al³ species,which inhibits root growth and damages root systems (Kochian, 1995,Annu. Rev. Plant Biol. 46: 237-260). As such, aluminum toxicity isaggravated by acid precipitation (e.g., acid rain). For example,contaminated soils in Brazil contain aluminum in amounts that range from11 to 124 g/Kg of soil. Another constraint on acid soils is phosphorous(P) deficiency, which is caused by P fixation with Al and Fe oxides onthe surface of clay minerals in acid soils. See Sanchez et al. 1997. In:Replenishing Soil Fertility in Africa, ed. R Buresh, P Sanchez, FCalhoun, pp. 1-46). Root damage reduces water and nutrient uptake andthus crop productivity. Low soil pH has been documented to reduce theyield on nearly 25% of the world's land presently under production. See,Wood, et al. (2000) in Pilot Analysis of Global Ecosystems:Agroecosystems (Int. Food Policy Res, Inst. And World Resources Inst.,Washington, D.C.), pp 45-54). Thus, there is a need to provide methodsand materials for increasing aluminum tolerance in plants.

SUMMARY

This document provides methods and materials related to plants havingincreased tolerance to aluminum (Al³⁺). For example, this documentprovides transgenic plants and plant cells having increased tolerance toaluminum, nucleic acids used to generate transgenic plants and plantcells having increased tolerance to aluminum, methods for making plantshaving increased tolerance to aluminum, and methods for making plantcells that can be used to generate plants having increased tolerance toaluminum. Such plants and plant cells can be grown in acidic soilscontaining elevated levels of Al³⁺, resulting in increased yield in suchsoils.

In one aspect, this document features a method of increasing plant yieldin soil containing elevated levels of Al³⁺. The method includes growinga plant comprising an exogenous nucleic acid on soil having an elevatedlevel of Al³⁺, wherein yield of the plant is increased as compared tothe corresponding yield of a control plant that does not comprise saidnucleic acid.

In some embodiments, the exogenous nucleic acid includes a regulatoryregion operably linked to a nucleotide sequence encoding a polypeptide,wherein the HMM bit score of the amino acid sequence of the polypeptideis greater than about 65, the HMM based on the amino acid sequencesdepicted in any one of FIGS. 1-4.

In some embodiments, the exogenous nucleic acid includes a regulatoryregion operably linked to a nucleotide sequence encoding a polypeptidehaving 90 percent or greater sequence identity to an amino acid sequenceset forth in SEQ ID NOs: 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15,17, 19, 20, 22, 24, 26, 28, 29, 31, 32, 33, 35, 36, 38, 40, 41, 42, 43,44, 45, 47, 48, 49, 50, 52, 54, 55, 56, 57, 58, 59, 61, 63, 64, 65, 66,67, 69, 70, 72, 73, 75, 77, 78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 93, 94, 96, 97, 98, 99, 100, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 113, 115, 117, 119, 120, 121, 122, 124, 125, 126, 127,129, 131, 132, 134, 136, 137, 138, 140, 142, 144, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 158, 159, 160, 162, 163, 164, 166,167, 169, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 190, 191, 192, 193, 194, 195, 197, 198,199, 200, 201, 202, 203, 205, 207, 209, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 223, 224, 225, 227, 228, 229, 230, 231, 232,233, 234, 235, 237, 239, 241, 243, 245, 246, 248, 250, 251, 253, 255,257, 258, 259, 261, 263, 265, 267, 269, 271, 273, 274, 276, 278, 280,281, 283, 284, 285, 287, 288, 290, 292, 293, 294, 296, 298, 300, 302,304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 329,331, 333, 335, 337, 338, 339, 341, 342, 343, 344, 345, 346, 347, 348,349, 350, 351, 353, 355, 357, 358, 360, 362, 364, 366, 368, 369, 371,373, 375, 376, 377, 378, 379, 380, 382, 384, 386, 388, 390, 392, 393,395, 396, 397, 398, 399, 400, 401, 402, 404, 405, 407, 409, 411, 413,414, 416, 418, 420, 421, 423, 424, 425, 426, 428, 429, 431, 432, 433,434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447,449, 451, 453, 454, 456, 457, 458, 459, 460, 461, 462, 463, 465, 467,468, 469, 470, 471, 472, 473, 474, 475, 476, 478, 480, 482, 484, 486,488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514,516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 535, 537, 539, 540,542, 544, 545, 547, 549, 551, 553, 555, 557, 558, 560, 562, 564, 568,570, 572, 574, 576, 578, 579, 580, 581, 583, 585, 587, 589, 591, 593,595, 597, 599, 601, 603, 605, 607, 608, 610, 612, 614, 616, 618, 620,622, 624, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647,649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 660, 662, 664, 666,668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 679, 681, 683, 685,686, 687, 689, 691, 693, 695, 696, 698, 699, 701, 702, 703, 704, 705,706, 707, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 721,722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735,736, 737, 738, 739, 740, 742, 744, 745, 746, 747, 749, 750, 752, 753,754, 755, 757, 759, 760, 761, 762, 764, 765, 766, 767, 768, 769, 770,771, and 772.

In some embodiments, the exogenous nucleic acid includes a regulatoryregion operably linked to a nucleotide sequence having 90 percent orgreater sequence identity to the nucleotide sequence set forth in SEQ IDNOs: 1, 6, 16, 18, 21, 23, 25, 27, 30, 34, 37, 39, 46, 51, 53, 60, 62,68, 71, 74, 76, 80, 92, 95, 101, 112, 114, 116, 118, 123, 128, 130, 133,135, 139, 141, 143, 145, 157, 161, 165, 168, 170, 189, 196, 204, 206,208, 210, 222, 226, 236, 238, 240, 242, 244, 247, 249, 252, 254, 256,260, 262, 264, 266, 268, 270, 272, 275, 277, 279, 282, 286, 289, 291,295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321,323, 325, 327, 330, 332, 334, 336, 340, 352, 354, 356, 359, 361, 363,365, 367, 370, 372, 374, 381, 383, 385, 387, 389, 391, 394, 403, 406,408, 410, 412, 415, 417, 419, 422, 427, 430, 448, 450, 452, 455, 464,466, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501,503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529,531, 533, 536, 538, 541, 543, 546, 548, 550, 552, 554, 556, 559, 561,563, 567, 569, 571, 573, 575, 577, 582, 584, 586, 588, 590, 592, 594,596, 598, 600, 602, 604, 606, 609, 611, 613, 615, 617, 619, 621, 623,626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 659, 661,663, 665, 667, 678, 680, 682, 684, 688, 690, 692, 694, 697, 700, 708,720, 741, 743, 748, 751, 756, 758, and 763.

In any of the methods described herein, the nucleotide sequence canencode a polypeptide having 95 percent or greater sequence identity tothe amino acid sequence set forth in SEQ ID NOs: 2, 3, 4, 5, 7, 8, 9,10, 11, 12, 13, 14, 15, 17, 19, 20, 22, 24, 26, 28, 29, 31, 32, 33, 35,36, 38, 40, 41, 42, 43, 44, 45, 47, 48, 49, 50, 52, 54, 55, 56, 57, 58,59, 61, 63, 64, 65, 66, 67, 69, 70, 72, 73, 75, 77, 78, 79, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 93, 94, 96, 97, 98, 99, 100, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 113, 115, 117, 119, 120, 121,122, 124, 125, 126, 127, 129, 131, 132, 134, 136, 137, 138, 140, 142,144, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 158, 159,160, 162, 163, 164, 166, 167, 169, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 190, 191, 192,193, 194, 195, 197, 198, 199, 200, 201, 202, 203, 205, 207, 209, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 223, 224, 225, 227,228, 229, 230, 231, 232, 233, 234, 235, 237, 239, 241, 243, 245, 246,248, 250, 251, 253, 255, 257, 258, 259, 261, 263, 265, 267, 269, 271,273, 274, 276, 278, 280, 281, 283, 284, 285, 287, 288, 290, 292, 293,294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320,322, 324, 326, 328, 329, 331, 333, 335, 337, 338, 339, 341, 342, 343,344, 345, 346, 347, 348, 349, 350, 351, 353, 355, 357, 358, 360, 362,364, 366, 368, 369, 371, 373, 375, 376, 377, 378, 379, 380, 382, 384,386, 388, 390, 392, 393, 395, 396, 397, 398, 399, 400, 401, 402, 404,405, 407, 409, 411, 413, 414, 416, 418, 420, 421, 423, 424, 425, 426,428, 429, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442,443, 444, 445, 446, 447, 449, 451, 453, 454, 456, 457, 458, 459, 460,461, 462, 463, 465, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476,478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504,506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532,534, 535, 537, 539, 540, 542, 544, 545, 547, 549, 551, 553, 555, 557,558, 560, 562, 564, 568, 570, 572, 574, 576, 578, 579, 580, 581, 583,585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 608, 610,612, 614, 616, 618, 620, 622, 624, 625, 627, 629, 631, 633, 635, 637,639, 641, 643, 645, 647, 649, 650, 651, 652, 653, 654, 655, 656, 657,658, 660, 662, 664, 666, 668, 669, 670, 671, 672, 673, 674, 675, 676,677, 679, 681, 683, 685, 686, 687, 689, 691, 693, 695, 696, 698, 699,701, 702, 703, 704, 705, 706, 707, 709, 710, 711, 712, 713, 714, 715,716, 717, 718, 719, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730,731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 742, 744, 745, 746,747, 749, 750, 752, 753, 754, 755, 757, 759, 760, 761, 762, 764, 765,766, 767, 768, 769, 770, 771, and 772.

In any of the methods described herein, the nucleotide sequence canencode a polypeptide having 98 percent or greater sequence identity tothe amino acid sequence set forth in SEQ ID NOs: 2, 3, 4, 5, 7, 8, 9,10, 11, 12, 13, 14, 15, 17, 19, 20, 22, 24, 26, 28, 29, 31, 32, 33, 35,36, 38, 40, 41, 42, 43, 44, 45, 47, 48, 49, 50, 52, 54, 55, 56, 57, 58,59, 61, 63, 64, 65, 66, 67, 69, 70, 72, 73, 75, 77, 78, 79, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 93, 94, 96, 97, 98, 99, 100, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 113, 115, 117, 119, 120, 121,122, 124, 125, 126, 127, 129, 131, 132, 134, 136, 137, 138, 140, 142,144, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 158, 159,160, 162, 163, 164, 166, 167, 169, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 190, 191, 192,193, 194, 195, 197, 198, 199, 200, 201, 202, 203, 205, 207, 209, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 223, 224, 225, 227,228, 229, 230, 231, 232, 233, 234, 235, 237, 239, 241, 243, 245, 246,248, 250, 251, 253, 255, 257, 258, 259, 261, 263, 265, 267, 269, 271,273, 274, 276, 278, 280, 281, 283, 284, 285, 287, 288, 290, 292, 293,294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320,322, 324, 326, 328, 329, 331, 333, 335, 337, 338, 339, 341, 342, 343,344, 345, 346, 347, 348, 349, 350, 351, 353, 355, 357, 358, 360, 362,364, 366, 368, 369, 371, 373, 375, 376, 377, 378, 379, 380, 382, 384,386, 388, 390, 392, 393, 395, 396, 397, 398, 399, 400, 401, 402, 404,405, 407, 409, 411, 413, 414, 416, 418, 420, 421, 423, 424, 425, 426,428, 429, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442,443, 444, 445, 446, 447, 449, 451, 453, 454, 456, 457, 458, 459, 460,461, 462, 463, 465, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476,478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504,506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532,534, 535, 537, 539, 540, 542, 544, 545, 547, 549, 551, 553, 555, 557,558, 560, 562, 564, 568, 570, 572, 574, 576, 578, 579, 580, 581, 583,585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 608, 610,612, 614, 616, 618, 620, 622, 624, 625, 627, 629, 631, 633, 635, 637,639, 641, 643, 645, 647, 649, 650, 651, 652, 653, 654, 655, 656, 657,658, 660, 662, 664, 666, 668, 669, 670, 671, 672, 673, 674, 675, 676,677, 679, 681, 683, 685, 686, 687, 689, 691, 693, 695, 696, 698, 699,701, 702, 703, 704, 705, 706, 707, 709, 710, 711, 712, 713, 714, 715,716, 717, 718, 719, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730,731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 742, 744, 745, 746,747, 749, 750, 752, 753, 754, 755, 757, 759, 760, 761, 762, 764, 765,766, 767, 768, 769, 770, 771, and 772.

In any of the methods described herein, the nucleotide sequence can have95 percent or greater (e.g., 98 percent or 99 percent or greater)sequence identity to the nucleotide sequence set forth in SEQ ID NOs: 1,6, 16, 18, 21, 23, 25, 27, 30, 34, 37, 39, 46, 51, 53, 60, 62, 68, 71,74, 76, 80, 92, 95, 101, 112, 114, 116, 118, 123, 128, 130, 133, 135,139, 141, 143, 145, 157, 161, 165, 168, 170, 189, 196, 204, 206, 208,210, 222, 226, 236, 238, 240, 242, 244, 247, 249, 252, 254, 256, 260,262, 264, 266, 268, 270, 272, 275, 277, 279, 282, 286, 289, 291, 295,297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323,325, 327, 330, 332, 334, 336, 340, 352, 354, 356, 359, 361, 363, 365,367, 370, 372, 374, 381, 383, 385, 387, 389, 391, 394, 403, 406, 408,410, 412, 415, 417, 419, 422, 427, 430, 448, 450, 452, 455, 464, 466,477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503,505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531,533, 536, 538, 541, 543, 546, 548, 550, 552, 554, 556, 559, 561, 563,567, 569, 571, 573, 575, 577, 582, 584, 586, 588, 590, 592, 594, 596,598, 600, 602, 604, 606, 609, 611, 613, 615, 617, 619, 621, 623, 626,628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 659, 661, 663,665, 667, 678, 680, 682, 684, 688, 690, 692, 694, 697, 700, 708, 720,741, 743, 748, 751, 756, 758, and 763.

In any of the methods described herein, the nucleotide sequence canencode the polypeptide set forth in SEQ ID NOs: 2, 3, 4, 5, 7, 8, 9, 10,11, 12, 13, 14, 15, 17, 19, 20, 22, 24, 26, 28, 29, 31, 32, 33, 35, 36,38, 40, 41, 42, 43, 44, 45, 47, 48, 49, 50, 52, 54, 55, 56, 57, 58, 59,61, 63, 64, 65, 66, 67, 69, 70, 72, 73, 75, 77, 78, 79, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 93, 94, 96, 97, 98, 99, 100, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 113, 115, 117, 119, 120, 121, 122,124, 125, 126, 127, 129, 131, 132, 134, 136, 137, 138, 140, 142, 144,146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 158, 159, 160,162, 163, 164, 166, 167, 169, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 190, 191, 192, 193,194, 195, 197, 198, 199, 200, 201, 202, 203, 205, 207, 209, 211, 212,213, 214, 215, 216, 217, 218, 219, 220, 221, 223, 224, 225, 227, 228,229, 230, 231, 232, 233, 234, 235, 237, 239, 241, 243, 245, 246, 248,250, 251, 253, 255, 257, 258, 259, 261, 263, 265, 267, 269, 271, 273,274, 276, 278, 280, 281, 283, 284, 285, 287, 288, 290, 292, 293, 294,296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322,324, 326, 328, 329, 331, 333, 335, 337, 338, 339, 341, 342, 343, 344,345, 346, 347, 348, 349, 350, 351, 353, 355, 357, 358, 360, 362, 364,366, 368, 369, 371, 373, 375, 376, 377, 378, 379, 380, 382, 384, 386,388, 390, 392, 393, 395, 396, 397, 398, 399, 400, 401, 402, 404, 405,407, 409, 411, 413, 414, 416, 418, 420, 421, 423, 424, 425, 426, 428,429, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443,444, 445, 446, 447, 449, 451, 453, 454, 456, 457, 458, 459, 460, 461,462, 463, 465, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 478,480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506,508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534,535, 537, 539, 540, 542, 544, 545, 547, 549, 551, 553, 555, 557, 558,560, 562, 564, 568, 570, 572, 574, 576, 578, 579, 580, 581, 583, 585,587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 608, 610, 612,614, 616, 618, 620, 622, 624, 625, 627, 629, 631, 633, 635, 637, 639,641, 643, 645, 647, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658,660, 662, 664, 666, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677,679, 681, 683, 685, 686, 687, 689, 691, 693, 695, 696, 698, 699, 701,702, 703, 704, 705, 706, 707, 709, 710, 711, 712, 713, 714, 715, 716,717, 718, 719, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731,732, 733, 734, 735, 736, 737, 738, 739, 740, 742, 744, 745, 746, 747,749, 750, 752, 753, 754, 755, 757, 759, 760, 761, 762, 764, 765, 766,767, 768, 769, 770, 771, and 772.

In any of the methods described herein, the method further can includeharvesting biomass from the plant.

In any of the methods described herein, the regulatory region can be apromoter. For example, the promoter can be selected from the groupconsisting of YP0092, PT0676, PT0708, PT0613, PT0672, PT0678, PT0688,PT0837, the napin promoter, the Arcelin-5 promoter, the phaseolin genepromoter, the soybean trypsin inhibitor promoter, the ACP promoter, thestearoyl-ACP desaturase gene promoter, the soybean a′ subunit ofβ-conglycinin promoter, the oleosin promoter, the 15 kD zein promoter,the 16 kD zein promoter, the 19 kD zein promoter, the 22 kD zeinpromoter, the 27 kD zein promoter, the Osgt-1 promoter, the beta-amylasegene promoter, the barley hordein gene promoter, p326, YP0144, YP190,p13879, YP0050, p32449, 21876, YP0158, YP0214, YP0380, PT0848, PT0633,the cauliflower mosaic virus (CaMV) ³⁵S promoter, the mannopine synthase(MAS) promoter, the 1′ or 2′ promoters derived from T-DNA ofAgrobacterium tumefaciens, the figwort mosaic virus 34S promoter, riceactin promoter, maize ubiquitin-1 promoter, ribulose-1,5-bisphosphatecarboxylase (RbcS) promoter, the pine cab6 promoter, the Cab-1 genepromoter from wheat, the CAB-1 promoter from spinach, the cab1R promoterfrom rice, the pyruvate orthophosphate dikinase (PPDK) promoter fromcorn, the tobacco Lhcb1*2 promoter, the Arabidopsis thaliana SUC2sucrose-H+ symporter promoter, and a thylakoid membrane protein promoterfrom spinach, and PT0585.

In any of the methods described herein, the plant containing theexogenous nucleic acid can have an improved growth rate relative to acorresponding plant that does not contain the nucleic acid.

In any of the methods described herein, the plant containing theexogenous nucleic acid can have improved vegetative growth relative to acorresponding plant that does not contain nucleic acid.

This document also features a method of increasing tolerance of a plantto elevated levels of aluminum. The method includes introducing into aplurality of plant cells an isolated nucleic acid comprising a nucleicacid sequence encoding a polypeptide having 90 percent or greatersequence identity to an amino acid sequence set forth in SEQ ID NOs: 2,3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 19, 20, 22, 24, 26, 28,29, 31, 32, 33, 35, 36, 38, 40, 41, 42, 43, 44, 45, 47, 48, 49, 50, 52,54, 55, 56, 57, 58, 59, 61, 63, 64, 65, 66, 67, 69, 70, 72, 73, 75, 77,78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 93, 94, 96, 97, 98,99, 100, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 113, 115,117, 119, 120, 121, 122, 124, 125, 126, 127, 129, 131, 132, 134, 136,137, 138, 140, 142, 144, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 158, 159, 160, 162, 163, 164, 166, 167, 169, 171, 172, 173,174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187,188, 190, 191, 192, 193, 194, 195, 197, 198, 199, 200, 201, 202, 203,205, 207, 209, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,223, 224, 225, 227, 228, 229, 230, 231, 232, 233, 234, 235, 237, 239,241, 243, 245, 246, 248, 250, 251, 253, 255, 257, 258, 259, 261, 263,265, 267, 269, 271, 273, 274, 276, 278, 280, 281, 283, 284, 285, 287,288, 290, 292, 293, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312,314, 316, 318, 320, 322, 324, 326, 328, 329, 331, 333, 335, 337, 338,339, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 353, 355,357, 358, 360, 362, 364, 366, 368, 369, 371, 373, 375, 376, 377, 378,379, 380, 382, 384, 386, 388, 390, 392, 393, 395, 396, 397, 398, 399,400, 401, 402, 404, 405, 407, 409, 411, 413, 414, 416, 418, 420, 421,423, 424, 425, 426, 428, 429, 431, 432, 433, 434, 435, 436, 437, 438,439, 440, 441, 442, 443, 444, 445, 446, 447, 449, 451, 453, 454, 456,457, 458, 459, 460, 461, 462, 463, 465, 467, 468, 469, 470, 471, 472,473, 474, 475, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496,498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524,526, 528, 530, 532, 534, 535, 537, 539, 540, 542, 544, 545, 547, 549,551, 553, 555, 557, 558, 560, 562, 564, 568, 570, 572, 574, 576, 578,579, 580, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603,605, 607, 608, 610, 612, 614, 616, 618, 620, 622, 624, 625, 627, 629,631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 650, 651, 652, 653,654, 655, 656, 657, 658, 660, 662, 664, 666, 668, 669, 670, 671, 672,673, 674, 675, 676, 677, 679, 681, 683, 685, 686, 687, 689, 691, 693,695, 696, 698, 699, 701, 702, 703, 704, 705, 706, 707, 709, 710, 711,712, 713, 714, 715, 716, 717, 718, 719, 721, 722, 723, 724, 725, 726,727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740,742, 744, 745, 746, 747, 749, 750, 752, 753, 754, 755, 757, 759, 760,761, 762, 764, 765, 766, 767, 768, 769, 770, 771, and 772; producing aplant from the plant cell; and growing the plant on soil having anelevated level of Al³⁺, wherein the plant has increased yield ascompared to that of a control plant that does not comprise the nucleicacid. The nucleic acid sequence encoding the polypeptide can be operablylinked to a regulatory region.

This document also features a method of increasing tolerance of a plantto elevated levels of aluminum. The method includes introducing into aplurality of plant cells an isolated nucleic acid comprising a nucleicacid sequence encoding a polypeptide having 90 percent or greatersequence identity to an amino acid sequence set forth in SEQ ID NOs: 2,3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 19, 20, 22, 24, 26, 28,29, 31, 32, 33, 35, 36, 38, 40, 41, 42, 43, 44, 45, 47, 48, 49, 50, 52,54, 55, 56, 57, 58, 59, 61, 63, 64, 65, 66, 67, 69, 70, 72, 73, 75, 77,78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 93, 94, 96, 97, 98,99, 100, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 113, 115,117, 119, 120, 121, 122, 124, 125, 126, 127, 129, 131, 132, 134, 136,137, 138, 140, 142, 144, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 158, 159, 160, 162, 163, 164, 166, 167, 169, 171, 172, 173,174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187,188, 190, 191, 192, 193, 194, 195, 197, 198, 199, 200, 201, 202, 203,205, 207, 209, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,223, 224, 225, 227, 228, 229, 230, 231, 232, 233, 234, 235, 237, 239,241, 243, 245, 246, 248, 250, 251, 253, 255, 257, 258, 259, 261, 263,265, 267, 269, 271, 273, 274, 276, 278, 280, 281, 283, 284, 285, 287,288, 290, 292, 293, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312,314, 316, 318, 320, 322, 324, 326, 328, 329, 331, 333, 335, 337, 338,339, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 353, 355,357, 358, 360, 362, 364, 366, 368, 369, 371, 373, 375, 376, 377, 378,379, 380, 382, 384, 386, 388, 390, 392, 393, 395, 396, 397, 398, 399,400, 401, 402, 404, 405, 407, 409, 411, 413, 414, 416, 418, 420, 421,423, 424, 425, 426, 428, 429, 431, 432, 433, 434, 435, 436, 437, 438,439, 440, 441, 442, 443, 444, 445, 446, 447, 449, 451, 453, 454, 456,457, 458, 459, 460, 461, 462, 463, 465, 467, 468, 469, 470, 471, 472,473, 474, 475, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496,498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524,526, 528, 530, 532, 534, 535, 537, 539, 540, 542, 544, 545, 547, 549,551, 553, 555, 557, 558, 560, 562, 564, 568, 570, 572, 574, 576, 578,579, 580, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603,605, 607, 608, 610, 612, 614, 616, 618, 620, 622, 624, 625, 627, 629,631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 650, 651, 652, 653,654, 655, 656, 657, 658, 660, 662, 664, 666, 668, 669, 670, 671, 672,673, 674, 675, 676, 677, 679, 681, 683, 685, 686, 687, 689, 691, 693,695, 696, 698, 699, 701, 702, 703, 704, 705, 706, 707, 709, 710, 711,712, 713, 714, 715, 716, 717, 718, 719, 721, 722, 723, 724, 725, 726,727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740,742, 744, 745, 746, 747, 749, 750, 752, 753, 754, 755, 757, 759, 760,761, 762, 764, 765, 766, 767, 768, 769, 770, 771, and 772; and selectinga plant produced from the plurality of plant cells that has an increasedtolerance to elevated Al³⁺ as compared to the tolerance in acorresponding control plant that does not comprise the isolated nucleicacid. The nucleic acid sequence encoding the polypeptide can be operablylinked to a regulatory region.

This document also features a plant cell that contains an exogenousnucleic acid, the exogenous nucleic acid comprising a regulatory regionoperably linked to a nucleotide sequence encoding a polypeptide, whereinthe HMM bit score of the amino acid sequence of the polypeptide isgreater than about 65, the HMM based on the amino acid sequence depictedin any one of FIGS. 1-4, and wherein a plant produced from the plantcell is tolerant of elevated soil levels of Al³⁺ as compared to that ofa control plant that does not comprise the nucleic acid, and wherein theplant is not tolerant of elevated saline levels.

This document also features a plant cell that contains an exogenousnucleic acid, the exogenous nucleic acid comprising a regulatory regionoperably linked to a nucleotide sequence encoding a polypeptide having90 percent or greater sequence identity to an amino acid sequence setforth in SEQ ID NOs: 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17,19, 20, 22, 24, 26, 28, 29, 31, 32, 33, 35, 36, 38, 40, 41, 42, 43, 44,45, 47, 48, 49, 50, 52, 54, 55, 56, 57, 58, 59, 61, 63, 64, 65, 66, 67,69, 70, 72, 73, 75, 77, 78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 93, 94, 96, 97, 98, 99, 100, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 113, 115, 117, 119, 120, 121, 122, 124, 125, 126, 127, 129,131, 132, 134, 136, 137, 138, 140, 142, 144, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 158, 159, 160, 162, 163, 164, 166, 167,169, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 188, 190, 191, 192, 193, 194, 195, 197, 198, 199,200, 201, 202, 203, 205, 207, 209, 211, 212, 213, 214, 215, 216, 217,218, 219, 220, 221, 223, 224, 225, 227, 228, 229, 230, 231, 232, 233,234, 235, 237, 239, 241, 243, 245, 246, 248, 250, 251, 253, 255, 257,258, 259, 261, 263, 265, 267, 269, 271, 273, 274, 276, 278, 280, 281,283, 284, 285, 287, 288, 290, 292, 293, 294, 296, 298, 300, 302, 304,306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 329, 331,333, 335, 337, 338, 339, 341, 342, 343, 344, 345, 346, 347, 348, 349,350, 351, 353, 355, 357, 358, 360, 362, 364, 366, 368, 369, 371, 373,375, 376, 377, 378, 379, 380, 382, 384, 386, 388, 390, 392, 393, 395,396, 397, 398, 399, 400, 401, 402, 404, 405, 407, 409, 411, 413, 414,416, 418, 420, 421, 423, 424, 425, 426, 428, 429, 431, 432, 433, 434,435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 449,451, 453, 454, 456, 457, 458, 459, 460, 461, 462, 463, 465, 467, 468,469, 470, 471, 472, 473, 474, 475, 476, 478, 480, 482, 484, 486, 488,490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516,518, 520, 522, 524, 526, 528, 530, 532, 534, 535, 537, 539, 540, 542,544, 545, 547, 549, 551, 553, 555, 557, 558, 560, 562, 564, 568, 570,572, 574, 576, 578, 579, 580, 581, 583, 585, 587, 589, 591, 593, 595,597, 599, 601, 603, 605, 607, 608, 610, 612, 614, 616, 618, 620, 622,624, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649,650, 651, 652, 653, 654, 655, 656, 657, 658, 660, 662, 664, 666, 668,669, 670, 671, 672, 673, 674, 675, 676, 677, 679, 681, 683, 685, 686,687, 689, 691, 693, 695, 696, 698, 699, 701, 702, 703, 704, 705, 706,707, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 721, 722,723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736,737, 738, 739, 740, 742, 744, 745, 746, 747, 749, 750, 752, 753, 754,755, 757, 759, 760, 761, 762, 764, 765, 766, 767, 768, 769, 770, 771,and 772, and wherein a plant produced from the plant cell is tolerant ofelevated soil levels of Al³⁺ as compared to that of a control plant thatdoes not comprise the nucleic acid, and wherein the plant is nottolerant of elevated saline levels.

This document also features a transgenic plant comprising a plant celldescribed herein, progeny of the plants that have increased tolerance toelevated Al³⁺ conditions as compared to that of a control plant thatdoes not comprise the nucleic acid, and wherein the progeny is nottolerant of elevated saline levels, seeds of the transgenic plants, andvegetative tissue of the transgenic plants, as well as food and feedproducts that include the vegetative tissue. The plant can be selectedfrom the group consisting of Panicum virgatum, Sorghum bicolor,Miscanthus giganteus, Saccharum sp., Populus balsamifera, Zea mays,Glycine max, Brassica napus, Triticum aestivum, Gossypium hirsutum,Oryza sativa, Helianthus annuus, Medicago sativa, Beta vulgaris, orPennisetum glaucum.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims. The word “comprising” inthe claims may be replaced by “consisting essentially of” or with“consisting of,” according to standard practice in patent law.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1D contain an alignment of the amino acid sequence ofCeresClone: 375578 (SEQ ID NO: 353) with homologous and/or orthologousamino acid sequences. In all the alignment figures shown herein, a dashin an aligned sequence represents a gap, i.e., a lack of an amino acidat that position. Identical amino acids or conserved amino acidsubstitutions among aligned sequences are identified by boxes. FIG. 1and the other alignment figures provided herein were generated using theprogram MUSCLE version 3.52.

FIGS. 2A-2C contain an alignment of the amino acid sequence ofCeresClone: 11684 (SEQ ID NO: 237) with homologous and/or orthologousamino acid sequences.

FIGS. 3A-3C contain an alignment of the amino acid sequence ofCeresClone: 24255 (SEQ ID NO: 451) with homologous and/or orthologousamino acid sequences.

FIGS. 4A-4G contain an alignment of the amino acid sequence ofCeresClone: 1752915 (SEQ ID NO: 2) with homologous and/or orthologousamino acid sequences.

FIG. 5 is a bar graph of whole plant weight (shoots plus roots) ofeither wild-type (WT) controls or transgenic switchgrass (Ceres Clone375578) treated with either pH 7.26 water (pH7); pH 4.0 water (pH4); orpH 4.0 water with aluminum (˜11 g AlCl₃/Kg soil; ˜0.00621 μM Al⁺³)(AlpH4). Aluminum toxicity had little to no effect on plant weight ofthe transgenic plants in comparison to control.

DETAILED DESCRIPTION

The invention features methods and materials related to modulatingaluminum tolerance in plants. In some embodiments, the plants may alsohave, for example, modulated levels of lignin, modified rootarchitecture, modified herbicide resistance, modified carotenoidbiosynthesis, or modulated cell wall content. The methods describedherein can include transforming a plant cell with a nucleic acidencoding an aluminum tolerance-modulating polypeptide, whereinexpression of the polypeptide results in a modulated level of aluminumtolerance. Plant cells produced using such methods can be grown toproduce plants having an increased tolerance to elevated levels ofaluminum. Such plants can have increased plant yield in soil containingelevated levels of Al³⁺.

I. Definitions

“Amino acid” refers to one of the twenty biologically occurring aminoacids and to synthetic amino acids, including D/L optical isomers.

“Cell type-preferential promoter” or “tissue-preferential promoter”refers to a promoter that drives expression preferentially in a targetcell type or tissue, respectively, but may also lead to sometranscription in other cell types or tissues as well.

“Control plant” refers to a plant that does not contain the exogenousnucleic acid present in a transgenic plant of interest, but otherwisehas the same or similar genetic background as such a transgenic plant. Asuitable control plant can be a non-transgenic wild type plant, anon-transgenic segregant from a transformation experiment, or atransgenic plant that contains an exogenous nucleic acid other than theexogenous nucleic acid of interest.

“Domains” are groups of substantially contiguous amino acids in apolypeptide that can be used to characterize protein families and/orparts of proteins. Such domains have a “fingerprint” or “signature” thatcan comprise conserved primary sequence, secondary structure, and/orthree-dimensional conformation. Generally, domains are correlated withspecific in vitro and/or in vivo activities. A domain can have a lengthof from 10 amino acids to 400 amino acids, e.g., 10 to 50 amino acids,or 25 to 100 amino acids, or 35 to 65 amino acids, or 35 to 55 aminoacids, or 45 to 60 amino acids, or 200 to 300 amino acids, or 300 to 400amino acids.

“Down-regulation” refers to regulation that decreases production ofexpression products (mRNA, polypeptide, or both) relative to basal ornative states.

“Exogenous” with respect to a nucleic acid indicates that the nucleicacid is part of a recombinant nucleic acid construct, or is not in itsnatural environment. For example, an exogenous nucleic acid can be asequence from one species introduced into another species, i.e., aheterologous nucleic acid. Typically, such an exogenous nucleic acid isintroduced into the other species via a recombinant nucleic acidconstruct. An exogenous nucleic acid can also be a sequence that isnative to an organism and that has been reintroduced into cells of thatorganism. An exogenous nucleic acid that includes a native sequence canoften be distinguished from the naturally occurring sequence by thepresence of non-natural sequences linked to the exogenous nucleic acid,e.g., non-native regulatory sequences flanking a native sequence in arecombinant nucleic acid construct. In addition, stably transformedexogenous nucleic acids typically are integrated at positions other thanthe position where the native sequence is found. It will be appreciatedthat an exogenous nucleic acid may have been introduced into aprogenitor and not into the cell under consideration. For example, atransgenic plant containing an exogenous nucleic acid can be the progenyof a cross between a stably transformed plant and a non-transgenicplant. Such progeny are considered to contain the exogenous nucleicacid.

“Expression” refers to the process of converting genetic information ofa polynucleotide into RNA through transcription, which is catalyzed byan enzyme, RNA polymerase, and into protein, through translation of mRNAon ribosomes.

“Heterologous polypeptide” as used herein refers to a polypeptide thatis not a naturally occurring polypeptide in a plant cell, e.g., atransgenic Panicum virgatum plant transformed with and expressing thecoding sequence for a nitrogen transporter polypeptide from a Zea maysplant.

“Isolated nucleic acid” as used herein includes a naturally-occurringnucleic acid, provided one or both of the sequences immediately flankingthat nucleic acid in its naturally-occurring genome is removed orabsent. Thus, an isolated nucleic acid includes, without limitation, anucleic acid that exists as a purified molecule or a nucleic acidmolecule that is incorporated into a vector or a virus. A nucleic acidexisting among hundreds to millions of other nucleic acids within, forexample, cDNA libraries, genomic libraries, or gel slices containing agenomic DNA restriction digest, is not to be considered an isolatednucleic acid.

“Modulation” of the level of aluminum tolerance refers to the change inthe level of the aluminum tolerance that is observed as a result ofexpression of, or transcription from, an exogenous nucleic acid in aplant cell and/or plant. The change in level is measured relative to thecorresponding level in control plants. Aluminum tolerance can beassessed by measuring root growth and/or plant height of plants grown inacidified soils containing elevated levels of Al³⁺. The concentration ofAl³⁺ considered to be elevated can be adjusted depending on the speciesbeing tested as plant species vary in their capacity to toleratealuminum. For example, rice is more tolerant of aluminum than sorghum.As such, to determine increased tolerance to aluminum in rice,concentrations greater than 160 μM Al³⁺ can be used. In sorghum,concentrations of around 27 μM Al³⁺ can be used. For switchgrass,concentrations of around 600 μM can be used.

“Nucleic acid” and “polynucleotide” are used interchangeably herein, andrefer to both RNA and DNA, including cDNA, genomic DNA, synthetic DNA,and DNA or RNA containing nucleic acid analogs. A nucleic acid can bedouble-stranded or single-stranded (i.e., a sense strand or an antisensestrand). Non-limiting examples of polynucleotides include genes, genefragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomalRNA, siRNA, micro-RNA, ribozymes, cDNA, recombinant polynucleotides,branched polynucleotides, nucleic acid probes and nucleic acid primers.A polynucleotide may contain unconventional or modified nucleotides.

“Operably linked” refers to the positioning of a regulatory region and asequence to be transcribed in a nucleic acid so that the regulatoryregion is effective for regulating transcription or translation of thesequence. For example, to operably link a coding sequence and aregulatory region, the translation initiation site of the translationalreading frame of the coding sequence is typically positioned between oneand about fifty nucleotides downstream of the regulatory region. Aregulatory region can, however, be positioned as much as about 5,000nucleotides upstream of the translation initiation site, or about 2,000nucleotides upstream of the transcription start site.

“Polypeptide” as used herein refers to a compound of two or more subunitamino acids, amino acid analogs, or other peptidomimetics, regardless ofpost-translational modification, e.g., phosphorylation or glycosylation.The subunits may be linked by peptide bonds or other bonds such as, forexample, ester or ether bonds. Full-length polypeptides, truncatedpolypeptides, point mutants, insertion mutants, splice variants,chimeric proteins, and fragments thereof are encompassed by thisdefinition.

“Progeny” includes descendants of a particular plant or plant line.Progeny of an instant plant include seeds formed on F₁, F₂, F₃, F₄, F₅,F₆ and subsequent generation plants, or seeds formed on BC₁, BC₂, BC₃,and subsequent generation plants, or seeds formed on F₁BC₁, F₁BC₂,F₁BC₃, and subsequent generation plants. The designation F₁ refers tothe progeny of a cross between two parents that are geneticallydistinct. The designations F₂, F₃, F₄, F₅ and F₆ refer to subsequentgenerations of self- or sib-pollinated progeny of an F₁ plant.

“Regulatory region” refers to a nucleic acid having nucleotide sequencesthat influence transcription or translation initiation and rate, andstability and/or mobility of a transcription or translation product.Regulatory regions include, without limitation, promoter sequences,enhancer sequences, response elements, protein recognition sites,inducible elements, protein binding sequences, 5′ and 3′ untranslatedregions (UTRs), transcriptional start sites, termination sequences,polyadenylation sequences, introns, and combinations thereof. Aregulatory region typically comprises at least a core (basal) promoter.A regulatory region also may include at least one control element, suchas an enhancer sequence, an upstream element or an upstream activationregion (UAR). For example, a suitable enhancer is a cis-regulatoryelement (−212 to −154) from the upstream region of the octopine synthase(ocs) gene. Fromm et al., The Plant Cell, 1:977-984 (1989).

“Up-regulation” refers to regulation that increases the level of anexpression product (mRNA, polypeptide, or both) relative to basal ornative states.

“Vector” refers to a replicon, such as a plasmid, phage, or cosmid, intowhich another DNA segment may be inserted so as to bring about thereplication of the inserted segment. Generally, a vector is capable ofreplication when associated with the proper control elements. The term“vector” includes cloning and expression vectors, as well as viralvectors and integrating vectors. An “expression vector” is a vector thatincludes a regulatory region.

II. Polypeptides

Polypeptides described herein include aluminum tolerance-modulatingpolypeptides. Aluminum tolerance-modulating polypeptides can beeffective to modulate (e.g., increase) aluminum tolerance when expressedin a plant or plant cell. Such polypeptides typically contain at leastone domain indicative of aluminum tolerance-modulating polypeptides, asdescribed in more detail herein. Aluminum tolerance-modulatingpolypeptides typically have an HMM bit score that is greater than 65 asdescribed in more detail herein. In some embodiments, aluminumtolerance-modulating polypeptides have greater than 80% identity to SEQID NOs: 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 19, 20, 22, 24,26, 28, 29, 31, 32, 33, 35, 36, 38, 40, 41, 42, 43, 44, 45, 47, 48, 49,50, 52, 54, 55, 56, 57, 58, 59, 61, 63, 64, 65, 66, 67, 69, 70, 72, 73,75, 77, 78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 93, 94, 96,97, 98, 99, 100, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 113,115, 117, 119, 120, 121, 122, 124, 125, 126, 127, 129, 131, 132, 134,136, 137, 138, 140, 142, 144, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 158, 159, 160, 162, 163, 164, 166, 167, 169, 171, 172,173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,187, 188, 190, 191, 192, 193, 194, 195, 197, 198, 199, 200, 201, 202,203, 205, 207, 209, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 223, 224, 225, 227, 228, 229, 230, 231, 232, 233, 234, 235, 237,239, 241, 243, 245, 246, 248, 250, 251, 253, 255, 257, 258, 259, 261,263, 265, 267, 269, 271, 273, 274, 276, 278, 280, 281, 283, 284, 285,287, 288, 290, 292, 293, 294, 296, 298, 300, 302, 304, 306, 308, 310,312, 314, 316, 318, 320, 322, 324, 326, 328, 329, 331, 333, 335, 337,338, 339, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 353,355, 357, 358, 360, 362, 364, 366, 368, 369, 371, 373, 375, 376, 377,378, 379, 380, 382, 384, 386, 388, 390, 392, 393, 395, 396, 397, 398,399, 400, 401, 402, 404, 405, 407, 409, 411, 413, 414, 416, 418, 420,421, 423, 424, 425, 426, 428, 429, 431, 432, 433, 434, 435, 436, 437,438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 449, 451, 453, 454,456, 457, 458, 459, 460, 461, 462, 463, 465, 467, 468, 469, 470, 471,472, 473, 474, 475, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494,496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522,524, 526, 528, 530, 532, 534, 535, 537, 539, 540, 542, 544, 545, 547,549, 551, 553, 555, 557, 558, 560, 562, 564, 568, 570, 572, 574, 576,578, 579, 580, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601,603, 605, 607, 608, 610, 612, 614, 616, 618, 620, 622, 624, 625, 627,629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 650, 651, 652,653, 654, 655, 656, 657, 658, 660, 662, 664, 666, 668, 669, 670, 671,672, 673, 674, 675, 676, 677, 679, 681, 683, 685, 686, 687, 689, 691,693, 695, 696, 698, 699, 701, 702, 703, 704, 705, 706, 707, 709, 710,711, 712, 713, 714, 715, 716, 717, 718, 719, 721, 722, 723, 724, 725,726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739,740, 742, 744, 745, 746, 747, 749, 750, 752, 753, 754, 755, 757, 759,760, 761, 762, 764, 765, 766, 767, 768, 769, 770, 771, or 772 asdescribed in more detail herein.

A. Domains Indicative of Aluminum Tolerance-Modulating Polypeptides

An aluminum tolerance-modulating polypeptide can contain a NAC domain,which is predicted to be characteristic of an aluminum tolerancemodulating polypeptide. SEQ ID NO: 2 sets forth the amino acid sequenceof a Panicum virgatum clone, identified herein as CeresClone: 1752915(SEQ ID NO:1), that is predicted to contain a NAC protein domain. Forexample, an aluminum tolerance-modulating polypeptide can comprise a NACprotein domain having 60 percent or greater (e.g., 65, 70, 75, 80, 85,90, 95, 97, 98, 99, or 100%) sequence identity to residues 14 to 139 ofSEQ ID NO: 2. In some embodiments, an aluminum tolerance-modulatingpolypeptide can comprise a NAC protein domain having 60 percent orgreater (e.g., 65, 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100%) sequenceidentity to the NAC protein domain of one or more of the polypeptidesset forth in SEQ ID NOs: 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17,19, 20, 22, 24, 26, 28, 29, 31, 32, 33, 35, 36, 38, 40, 41, 42, 43, 44,45, 47, 48, 49, 50, 52, 54, 55, 56, 57, 58, 59, 61, 63, 64, 65, 66, 67,69, 70, 72, 73, 75, 77, 78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 93, 94, 96, 97, 98, 99, 100, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 113, 115, 117, 119, 120, 121, 122, 124, 125, 126, 127, 129,131, 132, 134, 136, 137, 138, 140, 142, 144, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 158, 159, 160, 162, 163, 164, 166, 167,169, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 188, 190, 191, 192, 193, 194, 195, 197, 198, 199,200, 201, 202, 203, 205, 207, 209, 211, 212, 213, 214, 215, 216, 217,218, 219, 220, 221, 223, 224, 225, 227, 228, 229, 230, 231, 232, 233,234, and 235. The NAC protein domains of such sequences are set forth inthe Sequence Listing. The NAC domain (about 160 amino acids) wasidentified from the No apical meristem (NAM) protein domain, ATFL-2, andCUC2 (Cup-Shaped Cotyledon). See Ooka et al. 2003, DNA Res. 20: 239-247;Fang et al. 2008, Mol. Genet. Genomics 280: 547-563). The NAM proteindomain is characteristic of plant development proteins. NAM proteinshave a role in determining positions of meristems and primordial.Mutations in NAM result in the failure to develop a shoot apicalmeristem in petunia embryos. See, e.g., Souer, et al., Cell 85:159-170(1996).

An aluminum tolerance-modulating polypeptide can contain an AN1-likeZinc finger domain and an A20-like zinc finger domain, which arepredicted to be characteristic of an aluminum tolerance-modulatingpolypeptide. For example, SEQ ID NO: 237 sets forth the amino acidsequence of an Arabidopsis thaliana clone, identified herein asCeresClone: 11684 (SEQ ID NO: 236), that is predicted to containAN1-like and A20-like zinc finger domains. For example, an aluminumtolerance-modulating polypeptide can comprise an AN1-like Zinc fingerdomain having 60 percent or greater (e.g., 65, 70, 75, 80, 85, 90, 95,97, 98, 99, or 100%) sequence identity to residues 101 to 140 of SEQ IDNO: 237 and can comprise an A20-like Zinc finger domain having 60percent or greater (e.g., 65, 70, 75, 80, 85, 90, 95, 97, 98, 99, or100%) sequence identity to residues 25 to 47 of SEQ ID NO: 237. In someembodiments, an aluminum tolerance-modulating polypeptide can comprisean AN1-like Zinc finger domain and an A20-like zinc finger domain having60 percent or greater (e.g., 65, 70, 75, 80, 85, 90, 95, 97, 98, 99, or100%) sequence identity to the AN1-like and A20-like zinc finger domainsof one or more of the polypeptides set forth in SEQ ID NOs: 239, 241,243, 245, 246, 248, 250, 251, 253, 255, 257, 258, 259, 261, 263, 265,267, 269, 271, 273, 274, 276, 278, 280, 281, 283, 284, 285, 287, 288,290, 292, 293, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314,316, 318, 320, 322, 324, 326, 328, 329, 331, 333, 335, 337, 338, 339,341, 342, 343, 344, 345, 346, 347, 348, 349, 350, and 351. The AN1-likeand A20-like zinc finger domains of such sequences are set forth in theSequence Listing. The AN1-like zinc finger domain is a dimetal(zinc)-bound alpha/beta fold, with six conserved cysteines and twohistidines that can coordinate 2 zinc atoms. The A20-like zinc fingerdomain can be a ubiquitin binding domain that mediates self-association.Stress-associated proteins (SAPs) can have an AN1-like zinc fingerdomain and an A20-like zinc finger domain. See, e.g., Vij and Tyag, MolGenet Genomics, 276(6):565-75 (2006).

SEQ ID NO: 451 sets forth the amino acid sequence of an Arabidopsisthaliana clone, identified herein as CeresClone: 24255 (SEQ ID NO: 450),that also is predicted to contain AN1-like and A20-like zinc fingerdomains. For example, an aluminum tolerance-modulating polypeptide cancomprise an AN1-like Zinc finger domain having 60 percent or greater(e.g., 65, 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100%) sequenceidentity to residues 102 to 141 of SEQ ID NO: 451 and can comprise anA20-like Zinc finger domain having 60 percent or greater (e.g., 65, 70,75, 80, 85, 90, 95, 97, 98, 99, or 100%) sequence identity to residues14 to 36 of SEQ ID NO: 451. In some embodiments, an aluminumtolerance-modulating polypeptide can comprise an AN1-like Zinc fingerdomain and an A20-like zinc finger domain having 60 percent or greater(e.g., 65, 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100%) sequenceidentity to the AN1-like and A20-like zinc finger domains of one or moreof the polypeptides set forth in SEQ ID NOs: 453, 454, 456, 457, 458,459, 460, 461, 462, 463, 465, 467, 468, 469, 470, 471, 472, 473, 474,475, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500,502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528,530, 532, 534, 535, 537, 539, 540, 542, 544, 545, 547, 549, 551, 553,555, 557, 558, 560, 562, 564, 568, 570, 572, 574, 576, 578, 579, 580,581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607,608, 610, 612, 614, 616, 618, 620, 622, 624, 625, 627, 629, 631, 633,635, 637, 639, 641, 643, 645, 647, 649, 650, 651, 652, 653, 654, 655,656, 657, 658, 660, 662, 664, 666, 668, 669, 670, 671, 672, 673, 674,675, 676, 677, 679, 681, 683, 685, 686, 687, 689, 691, 693, 695, 696,698, 699, 701, 702, 703, 704, 705, 706, 707, 709, 710, 711, 712, 713,714, 715, 716, 717, 718, 719, 721, 722, 723, 724, 725, 726, 727, 728,729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 742, 744,745, 746, 747, 749, 750, 752, 753, 754, 755, 757, 759, 760, 761, 762,764, 765, 766, 767, 768, 769, 770, 771, and 772.

An aluminum tolerance-modulating polypeptide can contain an IQ domain,which is predicted to be characteristic of an aluminumtolerance-modulating polypeptide. SEQ ID NO: 353 sets forth the aminoacid sequence of a Zea mays clone, identified herein as CeresClone:375578 (SEQ ID NO: 352), that is predicted to contain an IQ domain. Forexample, an aluminum tolerance-modulating polypeptide can comprise an IQdomain having 60 percent or greater (e.g., 65, 70, 75, 80, 85, 90, 95,97, 98, 99, or 100%) sequence identity to residues 139 to 157 of SEQ IDNO: 353. In some embodiments, an aluminum tolerance-modulatingpolypeptide can comprise an IQ domain having 60 percent or greater(e.g., 65, 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100%) sequenceidentity to the IQ domain of one or more of the polypeptides set forthin SEQ ID NOs: 355, 357, 358, 360, 362, 364, 366, 368, 369, 371, 373,375, 376, 377, 378, 379, 380, 382, 384, 386, 388, 390, 392, 393, 395,396, 397, 398, 399, 400, 401, 402, 404, 405, 407, 409, 411, 413, 414,416, 418, 420, 421, 423, 424, 425, 426, 428, 429, 431, 432, 433, 434,435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, and449. The IQ domain is a consensus for calcium-independent binding ofcalmodulin, which is a calcium sensor and helps regulate events throughits interaction with a diverse group of cellular proteins. See Rhoadsand Friedberg, FASEB J., 11(5):331-40 (1997).

In some embodiments, an aluminum tolerance-modulating polypeptide istruncated at the amino- or carboxy-terminal end of a naturally occurringpolypeptide. A truncated polypeptide may retain certain domains of thenaturally occurring polypeptide while lacking others. Thus, lengthvariants that are up to 5 amino acids shorter or longer typicallyexhibit the aluminum tolerance-modulating activity of a truncatedpolypeptide. In some embodiments, a truncated polypeptide is a dominantnegative polypeptide. Expression in a plant of such a truncatedpolypeptide confers a difference in the level of aluminum tolerance of aplant as compared to the corresponding level of a control plant thatdoes not comprise the truncation.

B. Functional Homologs Identified by Reciprocal BLAST® (sequencesimilarity search)

In some embodiments, one or more functional homologs of a referencealuminum tolerance-modulating polypeptide defined by one or more of thePfam descriptions indicated above are suitable for use as aluminumtolerance-modulating polypeptides. A functional homolog is a polypeptidethat has sequence similarity to a reference polypeptide, and thatcarries out one or more of the biochemical or physiological function(s)of the reference polypeptide. A functional homolog and the referencepolypeptide may be natural occurring polypeptides, and the sequencesimilarity may be due to convergent or divergent evolutionary events. Assuch, functional homologs are sometimes designated in the literature ashomologs, or orthologs, or paralogs. Variants of a naturally occurringfunctional homolog, such as polypeptides encoded by mutants of a wildtype coding sequence, may themselves be functional homologs.

Functional homologs can also be created via site-directed mutagenesis ofthe coding sequence for an aluminum tolerance-modulating polypeptide, orby combining domains from the coding sequences for differentnaturally-occurring aluminum tolerance-modulating polypeptides (“domainswapping”). The term “functional homolog” is sometimes applied to thenucleic acid that encodes a functionally homologous polypeptide.Functional homologs can be identified by analysis of nucleotide andpolypeptide sequence alignments. For example, performing a query on adatabase of nucleotide or polypeptide sequences can identify homologs ofaluminum tolerance-modulating polypeptides. Sequence analysis caninvolve BLAST®, Reciprocal BLAST®, or PSI-BLAST® analysis ofnonredundant databases using an aluminum tolerance-modulatingpolypeptide amino acid sequence as the reference sequence. Amino acidsequence is, in some instances, deduced from the nucleotide sequence.Those polypeptides in the database that have greater than 40% sequenceidentity are candidates for further evaluation for suitability as analuminum tolerance-modulating polypeptide. Amino acid sequencesimilarity allows for conservative amino acid substitutions, such assubstitution of one hydrophobic residue for another or substitution ofone polar residue for another. If desired, manual inspection of suchcandidates can be carried out in order to narrow the number ofcandidates to be further evaluated. Manual inspection can be performedby selecting those candidates that appear to have domains present inaluminum tolerance-modulating polypeptides, e.g., conserved functionaldomains.

Conserved regions can be identified by locating a region within theprimary amino acid sequence of an aluminum tolerance-modulatingpolypeptide that is a repeated sequence, forms some secondary structure(e.g., helices and beta sheets), establishes positively or negativelycharged domains, or represents a protein motif or domain. See, e.g., thePfam web site describing consensus sequences for a variety of proteinmotifs and domains on the World Wide Web at sanger.ac.uk/Software/Pfam/and pfam.janelia.org/. A description of the information included at thePfam database is described in Sonnhammer et al., Nucl. Acids Res.,26:320-322 (1998); Sonnhammer et al., Proteins, 28:405-420 (1997); andBateman et al., Nucl. Acids Res., 27:260-262 (1999). Conserved regionsalso can be determined by aligning sequences of the same or relatedpolypeptides from closely related species. Closely related speciespreferably are from the same family. In some embodiments, alignment ofsequences from two different species is adequate.

Typically, polypeptides that exhibit at least about 40% amino acidsequence identity are useful to identify conserved regions. Conservedregions of related polypeptides exhibit at least 45% amino acid sequenceidentity (e.g., at least 50%, at least 60%, at least 70%, at least 80%,or at least 90% amino acid sequence identity). In some embodiments, aconserved region exhibits at least 92%, 94%, 96%, 98%, or 99% amino acidsequence identity.

Examples of amino acid sequences of functional homologs of thepolypeptide set forth in SEQ ID NO: 353 are provided in FIG. 1 and inthe Sequence Listing. Such functional homologs include, for example,CeresClone_106263 (SEQ ID NO: 355), CeresClone_335348 (SEQ ID NO: 357),GI_115440873 (SEQ ID NO: 358), CeresClone_826796 (SEQ ID NO: 360),CeresAnnot_1465047 (SEQ ID NO: 362), CeresClone_1919901 (SEQ ID NO:364), SEEDLINE:ME02064_CeresClone_375578 mutated (SEQ ID NO: 366),CeresClone_520008 (SEQ ID NO: 368), GI_7413581 (SEQ ID NO: 369),CeresClone_228069 (SEQ ID NO: 371), CeresClone_467508 (SEQ ID NO: 373),CeresClone_1829581 (SEQ ID NO: 375), GI_125550655 (SEQ ID NO: 376),GI_15231175 (SEQ ID NO: 377), GI_145357576 (SEQ ID NO: 378),GI_125528277 (SEQ ID NO: 379), GI_224032591 (SEQ ID NO: 380),CeresAnnot_8669409 (SEQ ID NO: 382), CeresClone_1901601 (SEQ ID NO:384), CeresClone_2034697 (SEQ ID NO: 386), CeresClone_1747444 (SEQ IDNO: 388), CeresClone_1998974 (SEQ ID NO: 390), CeresClone_1883040 (SEQID NO: 392), GI_326520123 (SEQ ID NO: 393), CeresClone_101697218 (SEQ IDNO: 395), GI_215701453 (SEQ ID NO: 396), GI_225449126 (SEQ ID NO: 397),GI_147809623 (SEQ ID NO: 398), GI_224109704 (SEQ ID NO: 399),GI_225439898 (SEQ ID NO: 400), GI_218196002 (SEQ ID NO: 401),GI_54306075 (SEQ ID NO: 402), CeresAnnot_1484880 (SEQ ID NO: 404),GI_224028605 (SEQ ID NO: 405), CeresAnnot_1528800 (SEQ ID NO: 407),CeresClone_1792902 (SEQ ID NO: 409), CeresClone_1806867 (SEQ ID NO:411), CeresClone_1727738 (SEQ ID NO: 413), GI_238007500 (SEQ ID NO:414), CeresAnnot_8724651 (SEQ ID NO: 416), CeresClone_1897134 (SEQ IDNO: 418), CeresClone_1859266 (SEQ ID NO: 420), GI_194696788 (SEQ ID NO:421), CeresAnnot_1475350 (SEQ ID NO: 423), GI_326490361 (SEQ ID NO:424), GI_224140165 (SEQ ID NO: 425), GI_255577665 (SEQ ID NO: 426),CeresClone_1886384 (SEQ ID NO: 428), GI_255568402 (SEQ ID NO: 429),CeresClone_1942871 (SEQ ID NO: 431), GI_326527367 (SEQ ID NO: 432),GI_297816500 (SEQ ID NO: 433), GI_297810377 (SEQ ID NO: 434),GI_302762472 (SEQ ID NO: 435), GI_302815615 (SEQ ID NO: 436),GI_326525172 (SEQ ID NO: 437), GI_116787496 (SEQ ID NO: 438),GI_224029961 (SEQ ID NO: 439), GI_312282973 (SEQ ID NO: 440),GI_15232741 (SEQ ID NO: 441), GI_302806862 (SEQ ID NO: 442),GI_302772817 (SEQ ID NO: 443), GI_240254538 (SEQ ID NO: 444),GI_297833734 (SEQ ID NO: 445), GI_2739366 (SEQ ID NO: 446), GI_297825811(SEQ ID NO: 447), and CeresClone_229668 (SEQ ID NO: 449). In some cases,a functional homolog of SEQ ID NO: 353 has an amino acid sequence withat least 45% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to theamino acid sequence set forth in SEQ ID NO: 353. In some cases, afunctional homolog of SEQ ID NO: 353 has an amino acid sequence with atleast 45% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to one ormore functional homologs of SEQ ID NO: 353 described above or set forthin the Sequence Listing. The polypeptide set forth in SEQ ID NO: 353, orthe functional homologs set forth above or in the Sequence Listing, canbe truncated at the N- or C-terminus. In one embodiment, a functionalhomolog of SEQ ID NO:353 contains an N-terminal truncation. For example,a functional homolog of SEQ ID NO: 353 such as SEQ ID NO:366 can includeamino acids aligning with residues 188 to 498 of SEQ ID NO: 353.

Examples of amino acid sequences of functional homologs of thepolypeptide set forth in SEQ ID NO: 237 are provided in FIG. 2 and inthe Sequence Listing. Such functional homologs include, for example,CeresClone_1847516 (SEQ ID NO: 239), CeresAnnot_8714481 (SEQ ID NO:241), CeresClone_1961986 (SEQ ID NO: 243), CeresClone_1464596 (SEQ IDNO: 245), GI_225450173 (SEQ ID NO: 246), CeresClone_532446 (SEQ ID NO:248), CeresAnnot_1493109 (SEQ ID NO: 250), GI_88866527 (SEQ ID NO: 251),CeresClone_1609861 (SEQ ID NO: 253), CeresClone_1620215 (SEQ ID NO:255), CeresClone_1732772 (SEQ ID NO: 257), GI_295148935 (SEQ ID NO:258), GI_125606142 (SEQ ID NO: 259), CeresClone_1040399 (SEQ ID NO:261), CeresClone_1093691 (SEQ ID NO: 263), CeresClone_974539 (SEQ ID NO:265), CeresClone_1832340 (SEQ ID NO: 267), CeresClone_1933211 (SEQ IDNO: 269), CeresClone_997558 (SEQ ID NO: 271), CeresAnnot_6041596 (SEQ IDNO: 273), GI_125564176 (SEQ ID NO: 274), CeresClone_1836064 (SEQ ID NO:276), CeresClone_1909693 (SEQ ID NO: 278), CeresClone_1765346 (SEQ IDNO: 280), GI_125546008 (SEQ ID NO: 281), CeresClone_1950900 (SEQ ID NO:283), GI_41350259 (SEQ ID NO: 284), GI_125588210 (SEQ ID NO: 285),CeresClone_1954395 (SEQ ID NO: 287), GI_18403408 (SEQ ID NO: 288),CeresClone_2010121 (SEQ ID NO: 290), CeresAnnot_6011486 (SEQ ID NO:292), GI_25082726 (SEQ ID NO: 293), GI_113196593 (SEQ ID NO: 294),CeresClone_1843021 (SEQ ID NO: 296), CeresClone_1931194 (SEQ ID NO:298), CeresClone_1850070 (SEQ ID NO: 300), CeresAnnot_6034955 (SEQ IDNO: 302), CeresAnnot_6119444 (SEQ ID NO: 304), CeresAnnot_6063956 (SEQID NO: 306), CeresAnnot_6015461 (SEQ ID NO: 308), CeresClone_696244 (SEQID NO: 310), CeresAnnot_1468973 (SEQ ID NO: 312), CeresClone_2019529(SEQ ID NO: 314), CeresClone_1492169 (SEQ ID NO: 316),CeresClone_1652996 (SEQ ID NO: 318), CeresClone_100861292 (SEQ ID NO:320), CeresClone_1875452 (SEQ ID NO: 322), CeresClone_296366 (SEQ ID NO:324), CeresClone_1468822 (SEQ ID NO: 326), CeresClone_1793946 (SEQ IDNO: 328), GI_297829802 (SEQ ID NO: 329), CeresClone_1094610 (SEQ ID NO:331), CeresClone_1084216 (SEQ ID NO: 333), CeresClone_100041169 (SEQ IDNO: 335), CeresClone_1619774 (SEQ ID NO: 337), GI_218685692 (SEQ ID NO:338), GI_295148937 (SEQ ID NO: 339), CeresClone_1783953 (SEQ ID NO:341), GI_242045152 (SEQ ID NO: 342), GI_223972713 (SEQ ID NO: 343),GI_326494504 (SEQ ID NO: 344), GI_326515226 (SEQ ID NO: 345),GI_163838754 (SEQ ID NO: 346), GI_163838756 (SEQ ID NO: 347),GI_163838762 (SEQ ID NO: 348), GI_326523409 (SEQ ID NO: 349),GI_255544230 (SEQ ID NO: 350), and GI_222822669 (SEQ ID NO: 351). Insome cases, a functional homolog of SEQ ID NO: 237 has an amino acidsequence with at least 45% sequence identity, e.g., 50%, 52%, 56%, 59%,61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to the amino acid sequence set forth in SEQ ID NO: 237. Insome cases, a functional homolog of SEQ ID NO: 237 has an amino acidsequence with at least 45% sequence identity, e.g., 50%, 52%, 56%, 59%,61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to one or more functional homologs of SEQ ID NO: 237 describedabove or set forth in the Sequence Listing.

Examples of amino acid sequences of functional homologs of thepolypeptide set forth in SEQ ID NO: 451 are provided in FIG. 3 and inthe Sequence Listing. Such functional homologs include, for example,CeresClone_1931889 (SEQ ID NO: 453), GI_225440926 (SEQ ID NO: 454),CeresClone_708446 (SEQ ID NO: 456), GI_255575635 (SEQ ID NO: 457),GI_193237563 (SEQ ID NO: 458), GI_222822693 (SEQ ID NO: 459),GI_224103059 (SEQ ID NO: 460), GI_302398693 (SEQ ID NO: 461),GI_222822667 (SEQ ID NO: 462), GI_116778998 (SEQ ID NO: 463),CeresClone_1748922 (SEQ ID NO: 465), CeresClone_1775820 (SEQ ID NO:467), GI_115468934 (SEQ ID NO: 468), GI_118424243 (SEQ ID NO: 469),GI_147783026 (SEQ ID NO: 470), GI_119367488 (SEQ ID NO: 471),GI_147860340 (SEQ ID NO: 472), GI_147792975 (SEQ ID NO: 473),GI_21359918 (SEQ ID NO: 474), GI_73951300 (SEQ ID NO: 475), GI_115477170(SEQ ID NO: 476), CeresClone_1798756 (SEQ ID NO: 478),CeresClone_1918424 (SEQ ID NO: 480), CeresClone_1929645 (SEQ ID NO:482), CeresClone_1845154 (SEQ ID NO: 484), CeresClone_1840507 (SEQ IDNO: 486), CeresClone_1853430 (SEQ ID NO: 488), CeresClone_1853189 (SEQID NO: 490), CeresClone_1808578 (SEQ ID NO: 492), CeresClone_1857508(SEQ ID NO: 494), CeresClone_1946245 (SEQ ID NO: 496),CeresClone_1974721 (SEQ ID NO: 498), CeresClone_1940441 (SEQ ID NO:500), CeresClone_1834813 (SEQ ID NO: 502), CeresClone_1848383 (SEQ IDNO: 504), CeresClone_1895219 (SEQ ID NO: 506), CeresClone_1866608 (SEQID NO: 508), CeresAnnot_1481252 (SEQ ID NO: 510), CeresAnnot_1474904(SEQ ID NO: 512), CeresAnnot_1450673 (SEQ ID NO: 514),CeresAnnot_1437933 (SEQ ID NO: 516), CeresAnnot_1448174 (SEQ ID NO:518), CeresAnnot_1447323 (SEQ ID NO: 520), CeresAnnot_1456578 (SEQ IDNO: 522), CeresAnnot_1491680 (SEQ ID NO: 524), CeresAnnot_1438851 (SEQID NO: 526), CeresAnnot_1440235 (SEQ ID NO: 528), CeresAnnot_1461453(SEQ ID NO: 530), CeresAnnot_1468974 (SEQ ID NO: 532),CeresAnnot_1474244 (SEQ ID NO: 534), GI_15231686 (SEQ ID NO: 535),CeresClone_5522 (SEQ ID NO: 537), CeresClone_30543 (SEQ ID NO: 539),GI_14596167 (SEQ ID NO: 540), CeresClone_14203 (SEQ ID NO: 542),CeresClone_13832 (SEQ ID NO: 544), GI_15221329 (SEQ ID NO: 545),CeresClone_975913 (SEQ ID NO: 547), CeresClone_967417 (SEQ ID NO: 549),CeresClone_974951 (SEQ ID NO: 551), CeresClone_958471 (SEQ ID NO: 553),CeresClone_979956 (SEQ ID NO: 555), CeresClone_962327 (SEQ ID NO: 557),GI_119720772 (SEQ ID NO: 558), CeresClone_1614593 (SEQ ID NO: 560),CeresClone_567184 (SEQ ID NO: 562), CeresClone_580467 (SEQ ID NO: 564),CeresClone_1021029 (SEQ ID NO: 568), CeresClone_1072112 (SEQ ID NO:570), CeresClone_481246 (SEQ ID NO: 572), CeresClone_1242573 (SEQ ID NO:574), CeresClone_537439 (SEQ ID NO: 576), CeresClone_547126 (SEQ ID NO:578), GI_92896423 (SEQ ID NO: 579), GI_124360119 (SEQ ID NO: 580),GI_122069751 (SEQ ID NO: 581), CeresClone_1030374 (SEQ ID NO: 583),CeresClone_1030540 (SEQ ID NO: 585), CeresClone_634261 (SEQ ID NO: 587),CeresClone_696374 (SEQ ID NO: 589), CeresClone_698573 (SEQ ID NO: 591),CeresClone_701370 (SEQ ID NO: 593), CeresClone_893059 (SEQ ID NO: 595),CeresClone_757664 (SEQ ID NO: 597), CeresClone_1387149 (SEQ ID NO: 599),CeresClone_1411371 (SEQ ID NO: 601), CeresClone_1380920 (SEQ ID NO:603), CeresClone_1360570 (SEQ ID NO: 605), CeresClone_1458498 (SEQ IDNO: 607), GI_60202503 (SEQ ID NO: 608), CeresClone_1554153 (SEQ ID NO:610), CeresClone_1362320 (SEQ ID NO: 612), CeresClone_1545291 (SEQ IDNO: 614), CeresClone_1589047 (SEQ ID NO: 616), CeresClone_101142707 (SEQID NO: 618), CeresClone_1433926 (SEQ ID NO: 620), CeresClone_1734621(SEQ ID NO: 622), CeresClone_1731531 (SEQ ID NO: 624), GI_5031281 (SEQID NO: 625), CeresClone_1911017 (SEQ ID NO: 627), CeresClone_1955228(SEQ ID NO: 629), CeresClone_1867313 (SEQ ID NO: 631),CeresClone_1955668 (SEQ ID NO: 633), CeresClone_1765871 (SEQ ID NO:635), CeresClone_1870976 (SEQ ID NO: 637), CeresClone_1990071 (SEQ IDNO: 639), CeresClone_1965620 (SEQ ID NO: 641), CeresClone_1995041 (SEQID NO: 643), CeresClone_2015820 (SEQ ID NO: 645), CeresClone_1724157(SEQ ID NO: 647), CeresClone_1724165 (SEQ ID NO: 649), GI_35187687 (SEQID NO: 650), GI_125556051 (SEQ ID NO: 651), GI_125561658 (SEQ ID NO:652), GI_115470773 (SEQ ID NO: 653), GI_115444813 (SEQ ID NO: 654),GI_115446479 (SEQ ID NO: 655), GI_115455855 (SEQ ID NO: 656),GI_115479855 (SEQ ID NO: 657), GI_57899571 (SEQ ID NO: 658),CeresAnnot_6035031 (SEQ ID NO: 660), CeresAnnot_6111586 (SEQ ID NO:662), CeresAnnot_6011488 (SEQ ID NO: 664), CeresAnnot_6063958 (SEQ IDNO: 666), CeresAnnot_6063957 (SEQ ID NO: 668),GI_38016527_Gossypium_barbadense (SEQ ID NO: 669),GI_75133829_Oryza_sativa (SEQ ID NO: 670),GI_125546011_Oryza_sativa_indica (SEQ ID NO: 671), GI_116778802_Picea(SEQ ID NO: 672), GI_116778893_Picea (SEQ ID NO: 673),GI_157849766_Brassica_rapa (SEQ ID NO: 674),GI_159474166_Chlamydomonas_reinhardtii (SEQ ID NO: 675),GI_168036656_Physcomitrella_patens (SEQ ID NO: 676),GI_168053490_Physcomitrella_patens (SEQ ID NO: 677),CeresClone_100879386_Zea mays (SEQ ID NO: 679),CeresClone_1738028_Musa_acuminata (SEQ ID NO: 681),CeresClone_2055733_Miscanthus (SEQ ID NO: 683), CeresClone_2056478Miscanthus (SEQ ID NO: 685), GI_297823437 (SEQ ID NO: 686), GI_297816570(SEQ ID NO: 687), CeresClone_1428270 (SEQ ID NO: 689),CeresClone_1447811 (SEQ ID NO: 691), CeresClone_1093477 (SEQ ID NO:693), CeresClone_1260056 (SEQ ID NO: 695), GI_255565591 (SEQ ID NO:696), CeresClone_1626661 (SEQ ID NO: 698), GI_222822665 (SEQ ID NO:699), CeresClone_1842119 (SEQ ID NO: 701), GI_222822659 (SEQ ID NO:702), GI_222822691 (SEQ ID NO: 703), GI_255544810 (SEQ ID NO: 704),GI_297847482 (SEQ ID NO: 705), GI_116791002 (SEQ ID NO: 706),GI_297809513 (SEQ ID NO: 707), CeresClone_100049493 (SEQ ID NO: 709),GI_222822685 (SEQ ID NO: 710), GI_301133546 (SEQ ID NO: 711),GI_225426659 (SEQ ID NO: 712), GI_297799728 (SEQ ID NO: 713),GI_15234402 (SEQ ID NO: 714), GI_254030287 (SEQ ID NO: 715),GI_222423788 (SEQ ID NO: 716), GI_222822679 (SEQ ID NO: 717),GI_222822653 (SEQ ID NO: 718), GI_302787300 (SEQ ID NO: 719),CeresClone_1955772 (SEQ ID NO: 721), GI_222822655 (SEQ ID NO: 722),GI_225435496 (SEQ ID NO: 723), GI_15235819 (SEQ ID NO: 724),GI_212275744 (SEQ ID NO: 725), GI_116787141 (SEQ ID NO: 726),GI_238575930 (SEQ ID NO: 727), GI_326515250 (SEQ ID NO: 728),GI_217075260 (SEQ ID NO: 729), GI_222622367 (SEQ ID NO: 730),GI_222822681 (SEQ ID NO: 731), GI_194692334 (SEQ ID NO: 732),GI_116791662 (SEQ ID NO: 733), GI_168007673 (SEQ ID NO: 734),GI_222822687 (SEQ ID NO: 735), GI_326499404 (SEQ ID NO: 736),GI_255628951 (SEQ ID NO: 737), GI_326496248 (SEQ ID NO: 738),GI_297849616 (SEQ ID NO: 739), GI_326489039 (SEQ ID NO: 740),CeresAnnot_8672029 (SEQ ID NO: 742), CeresClone_24253 (SEQ ID NO: 744),GI_297739707 (SEQ ID NO: 745), GI_217075452 (SEQ ID NO: 746),GI_168037934 (SEQ ID NO: 747), CeresClone_682858 (SEQ ID NO: 749),GI_222822689 (SEQ ID NO: 750), CeresAnnot_8644539 (SEQ ID NO: 752),GI_222822663 (SEQ ID NO: 753), GI_168018442 (SEQ ID NO: 754),GI_242081565 (SEQ ID NO: 755), CeresClone_1463779 (SEQ ID NO: 757),CeresAnnot_8713173 (SEQ ID NO: 759), GI_225455582 (SEQ ID NO: 760),GI_218189113 (SEQ ID NO: 761), GI_302755624 (SEQ ID NO: 762),CeresClone_1778054 (SEQ ID NO: 764), GI_302822895 (SEQ ID NO: 765),GI_255638082 (SEQ ID NO: 766), GI_297746352 (SEQ ID NO: 767),GI_255640538 (SEQ ID NO: 768), GI_13276697 (SEQ ID NO: 769),GI_255628269 (SEQ ID NO: 770), GI_297804888 (SEQ ID NO: 771), andGI_297736227 (SEQ ID NO: 772). In some cases, a functional homolog ofSEQ ID NO: 451 has an amino acid sequence with at least 45% sequenceidentity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence setforth in SEQ ID NO: 451. In some cases, a functional homolog of SEQ IDNO: 451 has an amino acid sequence with at least 45% sequence identity,e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,98%, or 99% sequence identity, to one or more functional homologs of SEQID NO: 451 described above or set forth in the Sequence Listing.

Examples of amino acid sequences of functional homologs of thepolypeptide set forth in SEQ ID NO: 2 are provided in FIG. 4 and in theSequence Listing. Such functional homologs include, for example,GI_239053200 (SEQ ID NO: 3), GI_242043390 (SEQ ID NO: 4), GI_4218535(SEQ ID NO: 5), CeresAnnot_1706255 (SEQ ID NO: 7), GI_82400207 (SEQ IDNO: 8), GI_326514348 (SEQ ID NO: 9), GI_225435840 (SEQ ID NO: 10),GI_155965519 (SEQ ID NO: 11), GI_116791569 (SEQ ID NO: 12), GI_82568708(SEQ ID NO: 13), GI_21105748 (SEQ ID NO: 14), GI_154362215 (SEQ ID NO:15), CeresClone_1804251 (SEQ ID NO: 17), CeresClone_219367 (SEQ ID NO:19), GI_226528637 (SEQ ID NO: 20), CeresClone_100906935 (SEQ ID NO: 22),CeresClone_1902485 (SEQ ID NO: 24), CeresClone_754901 (SEQ ID NO: 26),CeresClone_1804740 (SEQ ID NO: 28), GI_88770831 (SEQ ID NO: 29),CeresClone_100885834 (SEQ ID NO: 31), GI_125557725 (SEQ ID NO: 32),GI_115471229 (SEQ ID NO: 33), CeresAnnot_8631975 (SEQ ID NO: 35),GI_261349144 (SEQ ID NO: 36), CeresClone_261978 (SEQ ID NO: 38),CeresClone_1797688 (SEQ ID NO: 40), GI_300510868 (SEQ ID NO: 41),GI_51702426 (SEQ ID NO: 42), GI_219884691 (SEQ ID NO: 43), GI_292659262(SEQ ID NO: 44), GI_82400213 (SEQ ID NO: 45), CeresClone_1566599 (SEQ IDNO: 47), GI_6730938 (SEQ ID NO: 48), GI_125528153 (SEQ ID NO: 49),GI_185179439 (SEQ ID NO: 50), CeresClone_1810747 (SEQ ID NO: 52),CeresClone_736293 (SEQ ID NO: 54), GI_292659260 (SEQ ID NO: 55),GI_242059063 (SEQ ID NO: 56), GI_82400209 (SEQ ID NO: 57), GI_223975401(SEQ ID NO: 58), GI_138753440 (SEQ ID NO: 59), CeresClone_101131915 (SEQID NO: 61), CeresClone_100821085 (SEQ ID NO: 63), GI_302399001 (SEQ IDNO: 64), GI_225459603 (SEQ ID NO: 65), GI_302141792 (SEQ ID NO: 66),GI_125572421 (SEQ ID NO: 67), CeresClone_542522 (SEQ ID NO: 69),GI_195549547 (SEQ ID NO: 70), CeresClone_100961037 (SEQ ID NO: 72),GI_255640977 (SEQ ID NO: 73), CeresClone_1850651 (SEQ ID NO: 75),CeresAnnot_8462230 (SEQ ID NO: 77), GI_198400323 (SEQ ID NO: 78),GI_254034330 (SEQ ID NO: 79), CeresClone_100066176 (SEQ ID NO: 81),GI_206584339 (SEQ ID NO: 82), GI_224063050 (SEQ ID NO: 83), GI_312282805(SEQ ID NO: 84), GI_255558632 (SEQ ID NO: 85), GI_21105734 (SEQ ID NO:86), GI_116784797 (SEQ ID NO: 87), GI_187940307 (SEQ ID NO: 88),GI_184097796 (SEQ ID NO: 89), GI_222631638 (SEQ ID NO: 90), GI_116782512(SEQ ID NO: 91), CeresClone_1220729 (SEQ ID NO: 93), GI_14485513 (SEQ IDNO: 94), CeresClone_514804 (SEQ ID NO: 96), GI_155008462 (SEQ ID NO:97), GI_292659252 (SEQ ID NO: 98), GI_296044564 (SEQ ID NO: 99),GI_187940295 (SEQ ID NO: 100), CeresAnnot_8732688 (SEQ ID NO: 102),GI_302789271 (SEQ ID NO: 103), GI_206584337 (SEQ ID NO: 104),GI_326490045 (SEQ ID NO: 105), GI_31322570 (SEQ ID NO: 106),GI_168000027 (SEQ ID NO: 107), GI_297839605 (SEQ ID NO: 108),GI_62546185 (SEQ ID NO: 109), GI_147802535 (SEQ ID NO: 110),GI_302820063 (SEQ ID NO: 111), CeresClone_1769201 (SEQ ID NO: 113),CeresClone_477097 (SEQ ID NO: 115), CeresClone_1910025 (SEQ ID NO: 117),CeresAnnot_8669922 (SEQ ID NO: 119), GI_31322574 (SEQ ID NO: 120),GI_187940273 (SEQ ID NO: 121), GI_15223963 (SEQ ID NO: 122),CeresClone_100038358 (SEQ ID NO: 124), GI_148615629 (SEQ ID NO: 125),GI_225461361 (SEQ ID NO: 126), GI_255563837 (SEQ ID NO: 127),CeresClone_1727754 (SEQ ID NO: 129), CeresClone_1808216 (SEQ ID NO:131), GI_195549534 (SEQ ID NO: 132), CeresAnnot_1466775 (SEQ ID NO:134), CeresClone_23342 (SEQ ID NO: 136), GI_209171097 (SEQ ID NO: 137),GI_138753442 (SEQ ID NO: 138), CeresClone_1431867 (SEQ ID NO: 140),CeresClone_36478 (SEQ ID NO: 142), CeresClone_1864096 (SEQ ID NO: 144),CeresAnnot_1452119 (SEQ ID NO: 146), GI_302774106 (SEQ ID NO: 147),GI_171452372 (SEQ ID NO: 148), GI_297810999 (SEQ ID NO: 149),GI_21593134 (SEQ ID NO: 150), GI_125572883 (SEQ ID NO: 151),GI_242877145 (SEQ ID NO: 152), GI_125528621 (SEQ ID NO: 153),GI_115441473 (SEQ ID NO: 154), GI_31322566 (SEQ ID NO: 155),GI_224128213 (SEQ ID NO: 156), CeresClone_481915 (SEQ ID NO: 158),GI_31322580 (SEQ ID NO: 159), GI_311701727 (SEQ ID NO: 160),CeresClone_1434586 (SEQ ID NO: 162), GI_58013003 (SEQ ID NO: 163),GI_255586554 (SEQ ID NO: 164), CeresClone_1370400 (SEQ ID NO: 166),GI_312283489 (SEQ ID NO: 167), CeresAnnot_8453910 (SEQ ID NO: 169),CeresClone_1824711 (SEQ ID NO: 171), GI_57233056 (SEQ ID NO: 172),GI_167614348 (SEQ ID NO: 173), GI_296082607 (SEQ ID NO: 174),GI_62546187 (SEQ ID NO: 175), GI_62546189 (SEQ ID NO: 176), GI_31322568(SEQ ID NO: 177), GI_66275774 (SEQ ID NO: 178), GI_225438363 (SEQ ID NO:179), GI_124021383 (SEQ ID NO: 180), GI_224081060 (SEQ ID NO: 181),GI_255583865 (SEQ ID NO: 182), GI_217073174 (SEQ ID NO: 183),GI_168038326 (SEQ ID NO: 184), GI_295913643 (SEQ ID NO: 185),GI_31322578 (SEQ ID NO: 186), GI_118486672 (SEQ ID NO: 187), GI_15232604(SEQ ID NO: 188), CeresAnnot_8683724 (SEQ ID NO: 190), GI_187940303 (SEQID NO: 191), GI_311780299 (SEQ ID NO: 192), GI_297830130 (SEQ ID NO:193), GI_117586720 (SEQ ID NO: 194), GI_193237579 (SEQ ID NO: 195),CeresClone_1827014 (SEQ ID NO: 197), GI_31322576 (SEQ ID NO: 198),GI_255555947 (SEQ ID NO: 199), GI_125533643 (SEQ ID NO: 200),GI_118490007 (SEQ ID NO: 201), GI_21536744 (SEQ ID NO: 202),GI_302399025 (SEQ ID NO: 203), CeresClone_100046091 (SEQ ID NO: 205),CeresClone_20909 (SEQ ID NO: 207), CeresClone_1803889 (SEQ ID NO: 209),CeresAnnot_1456291 (SEQ ID NO: 211), GI_311976585 (SEQ ID NO: 212),GI_302399005 (SEQ ID NO: 213), GI_297744870 (SEQ ID NO: 214),GI_15223456 (SEQ ID NO: 215), GI_63252923 (SEQ ID NO: 216), GI_255636538(SEQ ID NO: 217), GI_113205414 (SEQ ID NO: 218), GI_113205453 (SEQ IDNO: 219), GI_255635167 (SEQ ID NO: 220), GI_206584345 (SEQ ID NO: 221),CeresAnnot_867355 (SEQ ID NO: 223), GI_242877103 (SEQ ID NO: 224),GI_302793560 (SEQ ID NO: 225), CeresClone_38344 (SEQ ID NO: 227),GI_31322582 (SEQ ID NO: 228), GI_115336269 (SEQ ID NO: 229),GI_311115260 (SEQ ID NO: 230), GI_168054147 (SEQ ID NO: 231),GI_311115262 (SEQ ID NO: 232), GI_168034283 (SEQ ID NO: 233),GI_15148914 (SEQ ID NO: 234), and GI_302767458 (SEQ ID NO: 235). In somecases, a functional homolog of SEQ ID NO: 2 has an amino acid sequencewith at least 45% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to theamino acid sequence set forth in SEQ ID NO: 2. In some cases, afunctional homolog of SEQ ID NO: 2 has an amino acid sequence with atleast 45% sequence identity, e.g., 50%, 52%, 56%, 59%, 61%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to one ormore functional homologs of SEQ ID NO: 2 described above or set forth inthe Sequence Listing.

The identification of conserved regions in an aluminumtolerance-modulating polypeptide facilitates production of variants ofaluminum tolerance-modulating polypeptides. Variants of aluminumtolerance-modulating polypeptides typically have 10 or fewerconservative amino acid substitutions within the primary amino acidsequence, e.g., 7 or fewer conservative amino acid substitutions, 5 orfewer conservative amino acid substitutions, or between 1 and 5conservative substitutions. A useful variant polypeptide can beconstructed based on one of the alignments set forth in FIG. 1, FIG. 2,FIG. 3, or FIG. 4, and/or homologs identified in the Sequence Listing.Such a polypeptide includes the conserved regions, arranged in the orderdepicted in the Figure from amino-terminal end to carboxy-terminal end.Such a polypeptide may also include zero, one, or more than one aminoacid in positions marked by dashes. When no amino acids are present atpositions marked by dashes, the length of such a polypeptide is the sumof the amino acid residues in all conserved regions. When amino acidsare present at a position marked by dashes, such a polypeptide has alength that is the sum of the amino acid residues in all conservedregions and all dashes.

C. Functional Homologs Identified by HMMER

In some embodiments, useful aluminum tolerance-modulating polypeptidesinclude those that fit a Hidden Markov Model based on the polypeptidesset forth in any one of FIGS. 1-4. A Hidden Markov Model (HMM) is astatistical model of a consensus sequence for a group of functionalhomologs. See, Durbin et al., Biological Sequence Analysis ProbabilisticModels of Proteins and Nucleic Acids, Cambridge University Press,Cambridge, UK (1998). An HMM is generated by the program HMMER 2.3.2with default program parameters, using the sequences of the group offunctional homologs as input. The multiple sequence alignment isgenerated by ProbCons (Do et al., Genome Res., 15(2):330-40 (2005))version 1.11 using a set of default parameters: -c, —consistency REPS of2; -ir, —iterative-refinement REPS of 100; -pre, —pre-training REPS of0. ProbCons is a public domain software program provided by StanfordUniversity.

The default parameters for building an HMM (hmmbuild) are as follows:the default “architecture prior” (archpri) used by MAP architectureconstruction is 0.85, and the default cutoff threshold (idlevel) used todetermine the effective sequence number is 0.62. HMMER 2.3.2 wasreleased Oct. 3, 2003 under a GNU general public license, and isavailable from various sources on the World Wide Web such ashmmer.janelia.org; hmmer.wustl.edu; and fr.com/hmmer232/. Hmmbuildoutputs the model as a text file.

The HMM for a group of functional homologs can be used to determine thelikelihood that a candidate aluminum tolerance-modulating polypeptidesequence is a better fit to that particular HMM than to a null HMMgenerated using a group of sequences that are not structurally orfunctionally related. The likelihood that a candidate polypeptidesequence is a better fit to an HMM than to a null HMM is indicated bythe HMM bit score, a number generated when the candidate sequence isfitted to the HMM profile using the HMMER hmmsearch program. Thefollowing default parameters are used when running hmmsearch: thedefault E-value cutoff (E) is 10.0, the default bit score cutoff (T) isnegative infinity, the default number of sequences in a database (Z) isthe real number of sequences in the database, the default E-value cutofffor the per-domain ranked hit list (domE) is infinity, and the defaultbit score cutoff for the per-domain ranked hit list (domT) is negativeinfinity. A high HMM bit score indicates a greater likelihood that thecandidate sequence carries out one or more of the biochemical orphysiological function(s) of the polypeptides used to generate the HMM.A high HMM bit score is at least 20, and often is higher. Slightvariations in the HMM bit score of a particular sequence can occur dueto factors such as the order in which sequences are processed foralignment by multiple sequence alignment algorithms such as the ProbConsprogram. Nevertheless, such HMM bit score variation is minor.

The aluminum tolerance-modulating polypeptides discussed below fit theindicated HMM with an HMM bit score greater than to 65 (e.g., greaterthan 70, 80, 90, 100, 120, 140, 200, 300, 400, 500, 600, 700, 800, 900,1000, 1500, or 2000). In some embodiments, the HMM bit score of analuminum tolerance-modulating polypeptide discussed below is about 50%,60%, 70%, 80%, 90%, or 95% of the HMM bit score of a functional homologprovided in the Sequence Listing of this application. In someembodiments, an aluminum tolerance-modulating polypeptide discussedbelow fits the indicated HMM with an HMM bit score greater than 210, andhas a domain indicative of an aluminum tolerance-modulating polypeptide.In some embodiments, an aluminum tolerance-modulating polypeptidediscussed below fits the indicated HMM with an HMM bit score greaterthan 210, and has 65% or greater sequence identity (e.g., 75%, 80%, 85%,90%, 95%, or 100% sequence identity) to an amino acid sequence shown inany one of FIGS. 1-4.

Examples of polypeptides are shown in the sequence listing that have HMMbit scores greater than 159 (e.g., greater than 160, 170, 180, 190, 200,225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550,575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900,925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200,1225, 1250, or 1260) when fitted to an HMM generated from the amino acidsequences set forth in FIG. 1 are identified in the Sequence Listing ofthis application. Such polypeptides include, for example, SEQ ID NOs:353, 355, 357, 358, 360, 362, 364, 366, 368, 369, 371, 373, 375, 376,377, 378, 379, 380, 382, 384, 386, 388, 390, 392, 393, 395, 396, 397,398, 399, 400, 401, 402, 404, 405, 407, 409, 411, 413, 414, 416, 418,420, 421, 423, 424, 425, 426, 428, 429, 431, 432, 433, 434, 435, 436,437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, and 449.

Examples of polypeptides are shown in the sequence listing that have HMMbit scores greater than 83 (e.g., greater than 85, 90, 95, 100, 110,120, 130, 140, 150, 175, 200, 225, 250, 275, or 300) when fitted to anHMM generated from the amino acid sequences set forth in FIG. 2 areidentified in the Sequence Listing of this application. Suchpolypeptides include, for example, SEQ ID NOs: 237, 239, 241, 243, 245,246, 248, 250, 251, 253, 255, 257, 258, 259, 261, 263, 265, 267, 269,271, 273, 274, 276, 278, 280, 281, 283, 284, 285, 287, 288, 290, 292,293, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318,320, 322, 324, 326, 328, 329, 331, 333, 335, 337, 338, 339, 341, 342,343, 344, 345, 346, 347, 348, 349, 350, and 351.

Examples of polypeptides are shown in the sequence listing that have HMMbit scores greater than 100 (e.g., greater than 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 410,420, or 430) when fitted to an HMM generated from the amino acidsequences set forth in FIG. 3 are identified in the Sequence Listing ofthis application. Such polypeptides include, for example, SEQ ID NOs:451, 453, 454, 456, 457, 458, 459, 460, 461, 462, 463, 465, 467, 468,469, 470, 471, 472, 473, 474, 475, 476, 478, 480, 482, 484, 486, 488,490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516,518, 520, 522, 524, 526, 528, 530, 532, 534, 535, 537, 539, 540, 542,544, 545, 547, 549, 551, 553, 555, 557, 558, 560, 562, 564, 568, 570,572, 574, 576, 578, 579, 580, 581, 583, 585, 587, 589, 591, 593, 595,597, 599, 601, 603, 605, 607, 608, 610, 612, 614, 616, 618, 620, 622,624, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649,650, 651, 652, 653, 654, 655, 656, 657, 658, 660, 662, 664, 666, 668,669, 670, 671, 672, 673, 674, 675, 676, 677, 679, 681, 683, 685, 686,687, 689, 691, 693, 695, 696, 698, 699, 701, 702, 703, 704, 705, 706,707, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 721, 722,723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736,737, 738, 739, 740, 742, 744, 745, 746, 747, 749, 750, 752, 753, 754,755, 757, 759, 760, 761, 762, 764, 765, 766, 767, 768, 769, 770, 771,and 772.

Examples of polypeptides are shown in the sequence listing that have HMMbit scores greater than 204 (e.g., greater than 210, 220, 230, 240, 250,260, 270, 280, 290, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525,550, 575, 600, 625, 650, 675, or 700) when fitted to an HMM generatedfrom the amino acid sequences set forth in FIG. 4 are identified in theSequence Listing of this application. Such polypeptides include, forexample, SEQ ID NOs: 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17,19, 20, 22, 24, 26, 28, 29, 31, 32, 33, 35, 36, 38, 40, 41, 42, 43, 44,45, 47, 48, 49, 50, 52, 54, 55, 56, 57, 58, 59, 61, 63, 64, 65, 66, 67,69, 70, 72, 73, 75, 77, 78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 93, 94, 96, 97, 98, 99, 100, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 113, 115, 117, 119, 120, 121, 122, 124, 125, 126, 127, 129,131, 132, 134, 136, 137, 138, 140, 142, 144, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 158, 159, 160, 162, 163, 164, 166, 167,169, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 188, 190, 191, 192, 193, 194, 195, 197, 198, 199,200, 201, 202, 203, 205, 207, 209, 211, 212, 213, 214, 215, 216, 217,218, 219, 220, 221, 223, 224, 225, 227, 228, 229, 230, 231, 232, 233,234, and 235.

D. Percent Identity

In some embodiments, an aluminum tolerance-modulating polypeptide has anamino acid sequence with at least 45% sequence identity, e.g., 50%, 52%,56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%sequence identity, to one of the amino acid sequences set forth in SEQID NOs: 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 19, 20, 22, 24,26, 28, 29, 31, 32, 33, 35, 36, 38, 40, 41, 42, 43, 44, 45, 47, 48, 49,50, 52, 54, 55, 56, 57, 58, 59, 61, 63, 64, 65, 66, 67, 69, 70, 72, 73,75, 77, 78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 93, 94, 96,97, 98, 99, 100, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 113,115, 117, 119, 120, 121, 122, 124, 125, 126, 127, 129, 131, 132, 134,136, 137, 138, 140, 142, 144, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 158, 159, 160, 162, 163, 164, 166, 167, 169, 171, 172,173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,187, 188, 190, 191, 192, 193, 194, 195, 197, 198, 199, 200, 201, 202,203, 205, 207, 209, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 223, 224, 225, 227, 228, 229, 230, 231, 232, 233, 234, 235, 237,239, 241, 243, 245, 246, 248, 250, 251, 253, 255, 257, 258, 259, 261,263, 265, 267, 269, 271, 273, 274, 276, 278, 280, 281, 283, 284, 285,287, 288, 290, 292, 293, 294, 296, 298, 300, 302, 304, 306, 308, 310,312, 314, 316, 318, 320, 322, 324, 326, 328, 329, 331, 333, 335, 337,338, 339, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 353,355, 357, 358, 360, 362, 364, 366, 368, 369, 371, 373, 375, 376, 377,378, 379, 380, 382, 384, 386, 388, 390, 392, 393, 395, 396, 397, 398,399, 400, 401, 402, 404, 405, 407, 409, 411, 413, 414, 416, 418, 420,421, 423, 424, 425, 426, 428, 429, 431, 432, 433, 434, 435, 436, 437,438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 449, 451, 453, 454,456, 457, 458, 459, 460, 461, 462, 463, 465, 467, 468, 469, 470, 471,472, 473, 474, 475, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494,496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522,524, 526, 528, 530, 532, 534, 535, 537, 539, 540, 542, 544, 545, 547,549, 551, 553, 555, 557, 558, 560, 562, 564, 568, 570, 572, 574, 576,578, 579, 580, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601,603, 605, 607, 608, 610, 612, 614, 616, 618, 620, 622, 624, 625, 627,629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 650, 651, 652,653, 654, 655, 656, 657, 658, 660, 662, 664, 666, 668, 669, 670, 671,672, 673, 674, 675, 676, 677, 679, 681, 683, 685, 686, 687, 689, 691,693, 695, 696, 698, 699, 701, 702, 703, 704, 705, 706, 707, 709, 710,711, 712, 713, 714, 715, 716, 717, 718, 719, 721, 722, 723, 724, 725,726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739,740, 742, 744, 745, 746, 747, 749, 750, 752, 753, 754, 755, 757, 759,760, 761, 762, 764, 765, 766, 767, 768, 769, 770, 771, and 772.Polypeptides having such a percent sequence identity often have a domainindicative of an aluminum tolerance-modulating polypeptide and/or havean HMM bit score that is greater than 65, as discussed above. Amino acidsequences of aluminum tolerance-modulating polypeptides having at least80% sequence identity to one of the amino acid sequences set forth inSEQ ID NOs: 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 19, 20, 22,24, 26, 28, 29, 31, 32, 33, 35, 36, 38, 40, 41, 42, 43, 44, 45, 47, 48,49, 50, 52, 54, 55, 56, 57, 58, 59, 61, 63, 64, 65, 66, 67, 69, 70, 72,73, 75, 77, 78, 79, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 93, 94,96, 97, 98, 99, 100, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,113, 115, 117, 119, 120, 121, 122, 124, 125, 126, 127, 129, 131, 132,134, 136, 137, 138, 140, 142, 144, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 158, 159, 160, 162, 163, 164, 166, 167, 169, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 190, 191, 192, 193, 194, 195, 197, 198, 199, 200, 201,202, 203, 205, 207, 209, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 223, 224, 225, 227, 228, 229, 230, 231, 232, 233, 234, 235,237, 239, 241, 243, 245, 246, 248, 250, 251, 253, 255, 257, 258, 259,261, 263, 265, 267, 269, 271, 273, 274, 276, 278, 280, 281, 283, 284,285, 287, 288, 290, 292, 293, 294, 296, 298, 300, 302, 304, 306, 308,310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 329, 331, 333, 335,337, 338, 339, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351,353, 355, 357, 358, 360, 362, 364, 366, 368, 369, 371, 373, 375, 376,377, 378, 379, 380, 382, 384, 386, 388, 390, 392, 393, 395, 396, 397,398, 399, 400, 401, 402, 404, 405, 407, 409, 411, 413, 414, 416, 418,420, 421, 423, 424, 425, 426, 428, 429, 431, 432, 433, 434, 435, 436,437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 449, 451, 453,454, 456, 457, 458, 459, 460, 461, 462, 463, 465, 467, 468, 469, 470,471, 472, 473, 474, 475, 476, 478, 480, 482, 484, 486, 488, 490, 492,494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520,522, 524, 526, 528, 530, 532, 534, 535, 537, 539, 540, 542, 544, 545,547, 549, 551, 553, 555, 557, 558, 560, 562, 564, 568, 570, 572, 574,576, 578, 579, 580, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599,601, 603, 605, 607, 608, 610, 612, 614, 616, 618, 620, 622, 624, 625,627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 650, 651,652, 653, 654, 655, 656, 657, 658, 660, 662, 664, 666, 668, 669, 670,671, 672, 673, 674, 675, 676, 677, 679, 681, 683, 685, 686, 687, 689,691, 693, 695, 696, 698, 699, 701, 702, 703, 704, 705, 706, 707, 709,710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 721, 722, 723, 724,725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738,739, 740, 742, 744, 745, 746, 747, 749, 750, 752, 753, 754, 755, 757,759, 760, 761, 762, 764, 765, 766, 767, 768, 769, 770, 771, and 772 areprovided in FIGS. 1-4 and in the Sequence Listing.

“Percent sequence identity” refers to the degree of sequence identitybetween any given reference sequence, e.g., SEQ ID NO: 1, and acandidate aluminum tolerance-modulating sequence. A candidate sequencetypically has a length that is from 80 percent to 200 percent of thelength of the reference sequence, e.g., 82, 85, 87, 89, 90, 93, 95, 97,99, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, or 200percent of the length of the reference sequence. A percent identity forany candidate nucleic acid or polypeptide relative to a referencenucleic acid or polypeptide can be determined as follows. A referencesequence (e.g., a nucleic acid sequence or an amino acid sequence) isaligned to one or more candidate sequences using the computer programClustalW (version 1.83, default parameters), which allows alignments ofnucleic acid or polypeptide sequences to be carried out across theirentire length (global alignment). Chema et al., Nucleic Acids Res.,31(13):3497-500 (2003).

ClustalW calculates the best match between a reference and one or morecandidate sequences, and aligns them so that identities, similaritiesand differences can be determined. Gaps of one or more residues can beinserted into a reference sequence, a candidate sequence, or both, tomaximize sequence alignments. For fast pairwise alignment of nucleicacid sequences, the following default parameters are used: word size: 2;window size: 4; scoring method: percentage; number of top diagonals: 4;and gap penalty: 5. For multiple alignment of nucleic acid sequences,the following parameters are used: gap opening penalty: 10.0; gapextension penalty: 5.0; and weight transitions: yes. For fast pairwisealignment of protein sequences, the following parameters are used: wordsize: 1; window size: 5; scoring method: percentage; number of topdiagonals: 5; gap penalty: 3. For multiple alignment of proteinsequences, the following parameters are used: weight matrix: blosum; gapopening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps:on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, andLys; residue-specific gap penalties: on. The ClustalW output is asequence alignment that reflects the relationship between sequences.ClustalW can be run, for example, at the Baylor College of MedicineSearch Launcher site on the World Wide Web(searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at theEuropean Bioinformatics Institute site on the World Wide Web(ebi.ac.uk/clustalw).

To determine percent identity of a candidate nucleic acid or amino acidsequence to a reference sequence, the sequences are aligned usingClustalW, the number of identical matches in the alignment is divided bythe length of the reference sequence, and the result is multiplied by100. It is noted that the percent identity value can be rounded to thenearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are roundeddown to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded upto 78.2.

In some cases, an aluminum tolerance-modulating polypeptide has an aminoacid sequence with at least 45% sequence identity, e.g., 50%, 52%, 56%,59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to the amino acid sequence set forth in SEQ ID NO: 353. Aminoacid sequences of polypeptides having greater than 45% sequence identityto the polypeptide set forth in SEQ ID NO: 353 are provided in FIG. 1and in the Sequence Listing.

In some cases, an aluminum tolerance-modulating polypeptide has an aminoacid sequence with at least 45% sequence identity, e.g., 50%, 52%, 56%,59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to the amino acid sequence set forth in SEQ ID NO: 237. Aminoacid sequences of polypeptides having greater than 45% sequence identityto the polypeptide set forth in SEQ ID NO: 237 are provided in FIG. 2and in the Sequence Listing.

In some cases, an aluminum tolerance-modulating polypeptide has an aminoacid sequence with at least 45% sequence identity, e.g., 50%, 52%, 56%,59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to the amino acid sequence set forth in SEQ ID NO: 451. Aminoacid sequences of polypeptides having greater than 45% sequence identityto the polypeptide set forth in SEQ ID NO: 451 are provided in FIG. 3and in the Sequence Listing.

In some cases, an aluminum tolerance-modulating polypeptide has an aminoacid sequence with at least 45% sequence identity, e.g., 50%, 52%, 56%,59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequenceidentity, to the amino acid sequence set forth in SEQ ID NO: 2. Aminoacid sequences of polypeptides having greater than 45% sequence identityto the polypeptide set forth in SEQ ID NO: 2 are provided in FIG. 4 andin the Sequence Listing.

E. Other Sequences

It should be appreciated that an aluminum tolerance-modulatingpolypeptide can include additional amino acids that are not involved inaluminum tolerance modulation, and thus such a polypeptide can be longerthan would otherwise be the case. For example, an aluminumtolerance-modulating polypeptide can include a purification tag, achloroplast transit peptide, a mitochondrial transit peptide, anamyloplast peptide, or a leader sequence added to the amino or carboxyterminus. In some embodiments, an aluminum tolerance-modulatingpolypeptide includes an amino acid sequence that functions as areporter, e.g., a green fluorescent protein or yellow fluorescentprotein.

III. Nucleic Acids

Nucleic acids described herein include nucleic acids that are effectiveto modulate aluminum tolerance levels when transcribed in a plant orplant cell. Such nucleic acids include, without limitation, those thatencode an aluminum tolerance-modulating polypeptide and those that canbe used to inhibit expression of an aluminum tolerance-modulatingpolypeptide via a nucleic acid based method.

A. Nucleic Acids Encoding Aluminum Tolerance-Modulating Polypeptides

Nucleic acids encoding aluminum tolerance-modulating polypeptides aredescribed herein. Examples of such nucleic acids include SEQ ID NOs: 1,6, 16, 18, 21, 23, 25, 27, 30, 34, 37, 39, 46, 51, 53, 60, 62, 68, 71,74, 76, 80, 92, 95, 101, 112, 114, 116, 118, 123, 128, 130, 133, 135,139, 141, 143, 145, 157, 161, 165, 168, 170, 189, 196, 204, 206, 208,210, 222, 226, 236, 238, 240, 242, 244, 247, 249, 252, 254, 256, 260,262, 264, 266, 268, 270, 272, 275, 277, 279, 282, 286, 289, 291, 295,297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323,325, 327, 330, 332, 334, 336, 340, 352, 354, 356, 359, 361, 363, 365,367, 370, 372, 374, 381, 383, 385, 387, 389, 391, 394, 403, 406, 408,410, 412, 415, 417, 419, 422, 427, 430, 448, 450, 452, 455, 464, 466,477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503,505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531,533, 536, 538, 541, 543, 546, 548, 550, 552, 554, 556, 559, 561, 563,567, 569, 571, 573, 575, 577, 582, 584, 586, 588, 590, 592, 594, 596,598, 600, 602, 604, 606, 609, 611, 613, 615, 617, 619, 621, 623, 626,628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 659, 661, 663,665, 667, 678, 680, 682, 684, 688, 690, 692, 694, 697, 700, 708, 720,741, 743, 748, 751, 756, 758, and 763 as described in more detail below.A nucleic acid also can be a fragment that is at least 40% (e.g., atleast 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99%) of the lengthof the full-length nucleic acid set forth in SEQ ID NOs: 1, 6, 16, 18,21, 23, 25, 27, 30, 34, 37, 39, 46, 51, 53, 60, 62, 68, 71, 74, 76, 80,92, 95, 101, 112, 114, 116, 118, 123, 128, 130, 133, 135, 139, 141, 143,145, 157, 161, 165, 168, 170, 189, 196, 204, 206, 208, 210, 222, 226,236, 238, 240, 242, 244, 247, 249, 252, 254, 256, 260, 262, 264, 266,268, 270, 272, 275, 277, 279, 282, 286, 289, 291, 295, 297, 299, 301,303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 330,332, 334, 336, 340, 352, 354, 356, 359, 361, 363, 365, 367, 370, 372,374, 381, 383, 385, 387, 389, 391, 394, 403, 406, 408, 410, 412, 415,417, 419, 422, 427, 430, 448, 450, 452, 455, 464, 466, 477, 479, 481,483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509,511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 536, 538,541, 543, 546, 548, 550, 552, 554, 556, 559, 561, 563, 567, 569, 571,573, 575, 577, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602,604, 606, 609, 611, 613, 615, 617, 619, 621, 623, 626, 628, 630, 632,634, 636, 638, 640, 642, 644, 646, 648, 659, 661, 663, 665, 667, 678,680, 682, 684, 688, 690, 692, 694, 697, 700, 708, 720, 741, 743, 748,751, 756, 758, and 763.

An aluminum tolerance-modulating nucleic acid can comprise thenucleotide sequence set forth in SEQ ID NO:352. Alternatively, analuminum tolerance-modulating nucleic acid can be a variant of thenucleic acid having the nucleotide sequence set forth in SEQ ID NO: 352.For example, an aluminum tolerance-modulating nucleic acid can have anucleotide sequence with at least 80% sequence identity, e.g., 81%, 85%,90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleotide sequenceset forth in SEQ ID NO: 352.

An aluminum tolerance-modulating nucleic acid can comprise thenucleotide sequence set forth in SEQ ID NO: 236. Alternatively, analuminum tolerance-modulating nucleic acid can be a variant of thenucleic acid having the nucleotide sequence set forth in SEQ ID NO: 236.For example, an aluminum tolerance-modulating nucleic acid can have anucleotide sequence with at least 80% sequence identity, e.g., 81%, 85%,90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleotide sequenceset forth in SEQ ID NO: 236.

An aluminum tolerance-modulating nucleic acid can comprise thenucleotide sequence set forth in SEQ ID NO: 450. Alternatively, analuminum tolerance-modulating nucleic acid can be a variant of thenucleic acid having the nucleotide sequence set forth in SEQ ID NO: 450.For example, an aluminum tolerance-modulating nucleic acid can have anucleotide sequence with at least 80% sequence identity, e.g., 81%, 85%,90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleotide sequenceset forth in SEQ ID NO: 450.

An aluminum tolerance-modulating nucleic acid can comprise thenucleotide sequence set forth in SEQ ID NO: 1. Alternatively, analuminum tolerance-modulating nucleic acid can be a variant of thenucleic acid having the nucleotide sequence set forth in SEQ ID NO: 1.For example, an aluminum tolerance-modulating nucleic acid can have anucleotide sequence with at least 80% sequence identity, e.g., 81%, 85%,90%, 95%, 97%, 98%, or 99% sequence identity, to the nucleotide sequenceset forth in SEQ ID NO: 1.

Isolated nucleic acid molecules can be produced by standard techniques.For example, polymerase chain reaction (PCR) techniques can be used toobtain an isolated nucleic acid containing a nucleotide sequencedescribed herein. PCR can be used to amplify specific sequences from DNAas well as RNA, including sequences from total genomic DNA or totalcellular RNA. Various PCR methods are described, for example, in PCRPrimer: A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold SpringHarbor Laboratory Press, 1995. Generally, sequence information from theends of the region of interest or beyond is employed to designoligonucleotide primers that are identical or similar in sequence toopposite strands of the template to be amplified. Various PCR strategiesalso are available by which site-specific nucleotide sequencemodifications can be introduced into a template nucleic acid. Isolatednucleic acids also can be chemically synthesized, either as a singlenucleic acid molecule (e.g., using automated DNA synthesis in the 3′ to5′ direction using phosphoramidite technology) or as a series ofoligonucleotides. For example, one or more pairs of longoligonucleotides (e.g., >100 nucleotides) can be synthesized thatcontain the desired sequence, with each pair containing a short segmentof complementarity (e.g., about 15 nucleotides) such that a duplex isformed when the oligonucleotide pair is annealed. DNA polymerase is usedto extend the oligonucleotides, resulting in a single, double-strandednucleic acid molecule per oligonucleotide pair, which then can beligated into a vector. Isolated nucleic acids of the invention also canbe obtained by mutagenesis of, e.g., a naturally occurring DNA.

B. Use of Nucleic Acids to Modulate Expression of Polypeptides

i. Expression of an Aluminum Tolerance-Modulating Polypeptide

A nucleic acid encoding one of the aluminum tolerance-modulatingpolypeptides described herein can be used to express the polypeptide ina plant species of interest, typically by transforming a plant cell witha nucleic acid having the coding sequence for the polypeptide operablylinked in sense orientation to one or more regulatory regions. It willbe appreciated that because of the degeneracy of the genetic code, anumber of nucleic acids can encode a particular aluminumtolerance-modulating polypeptide; i.e., for many amino acids, there ismore than one nucleotide triplet that serves as the codon for the aminoacid. Thus, codons in the coding sequence for a given aluminumtolerance-modulating polypeptide can be modified such that optimalexpression in a particular plant species is obtained, using appropriatecodon bias tables for that species.

In some cases, expression of an aluminum tolerance-modulatingpolypeptide inhibits one or more functions of an endogenous polypeptide.For example, a nucleic acid that encodes a dominant negative polypeptidecan be used to inhibit protein function. A dominant negative polypeptidetypically is mutated or truncated relative to an endogenous wild typepolypeptide, and its presence in a cell inhibits one or more functionsof the wild type polypeptide in that cell, i.e., the dominant negativepolypeptide is genetically dominant and confers a loss of function. Themechanism by which a dominant negative polypeptide confers such aphenotype can vary but often involves a protein-protein interaction or aprotein-DNA interaction. For example, a dominant negative polypeptidecan be an enzyme that is truncated relative to a native wild typeenzyme, such that the truncated polypeptide retains domains involved inbinding a first protein but lacks domains involved in binding a secondprotein. The truncated polypeptide is thus unable to properly modulatethe activity of the second protein. See, e.g., US 2007/0056058. Asanother example, a point mutation that results in a non-conservativeamino acid substitution in a catalytic domain can result in a dominantnegative polypeptide. See, e.g., US 2005/032221. As another example, adominant negative polypeptide can be a transcription factor that istruncated relative to a native wild type transcription factor, such thatthe truncated polypeptide retains the DNA binding domain(s) but lacksthe activation domain(s). Such a truncated polypeptide can inhibit thewild type transcription factor from binding DNA, thereby inhibitingtranscription activation.

ii. Inhibition of Expression of an Aluminum Tolerance-ModulatingPolypeptide

Polynucleotides and recombinant constructs described herein can be usedto inhibit expression of an aluminum tolerance-modulating polypeptide ina plant species of interest. See, e.g., Matzke and Birchler, NatureReviews Genetics 6:24-35 (2005); Akashi et al., Nature Reviews Mol.Cell. Biology 6:413-422 (2005); Mittal, Nature Reviews Genetics5:355-365 (2004); and Nature Reviews RNA interference collection,October 2005 on the World Wide Web at nature.com/reviews/focus/mai. Anumber of nucleic acid based methods, including antisense RNA, ribozymedirected RNA cleavage, post-transcriptional gene silencing (PTGS), e.g.,RNA interference (RNAi), and transcriptional gene silencing (TGS) areknown to inhibit gene expression in plants. Suitable polynucleotidesinclude full-length nucleic acids encoding aluminum tolerance-modulatingpolypeptides or fragments of such full-length nucleic acids. In someembodiments, a complement of the full-length nucleic acid or a fragmentthereof can be used. Typically, a fragment is at least 10 nucleotides,e.g., at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 30, 35, 40, 50, 80, 100, 200, 500 nucleotides or more.Generally, higher homology can be used to compensate for the use of ashorter sequence.

Antisense technology is one well-known method. In this method, a nucleicacid of a gene to be repressed is cloned and operably linked to aregulatory region and a transcription termination sequence so that theantisense strand of RNA is transcribed. The recombinant construct isthen transformed into plants, as described herein, and the antisensestrand of RNA is produced. The nucleic acid need not be the entiresequence of the gene to be repressed, but typically will besubstantially complementary to at least a portion of the sense strand ofthe gene to be repressed.

In another method, a nucleic acid can be transcribed into a ribozyme, orcatalytic RNA, that affects expression of an mRNA. See, U.S. Pat. No.6,423,885. Ribozymes can be designed to specifically pair with virtuallyany target RNA and cleave the phosphodiester backbone at a specificlocation, thereby functionally inactivating the target RNA. Heterologousnucleic acids can encode ribozymes designed to cleave particular mRNAtranscripts, thus preventing expression of a polypeptide. Hammerheadribozymes are useful for destroying particular mRNAs, although variousribozymes that cleave mRNA at site-specific recognition sequences can beused. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target RNA contains a 5′-UG-3′nucleotide sequence. The construction and production of hammerheadribozymes is known in the art. See, for example, U.S. Pat. No. 5,254,678and WO 02/46449 and references cited therein. Hammerhead ribozymesequences can be embedded in a stable RNA such as a transfer RNA (tRNA)to increase cleavage efficiency in vivo. Perriman et al., Proc. Natl.Acad. Sci. USA, 92(13):6175-6179 (1995); de Feyter and Gaudron, Methodsin Molecular Biology, Vol. 74, Chapter 43, “Expressing Ribozymes inPlants”, Edited by Turner, P.C., Humana Press Inc., Totowa, N.J. RNAendoribonucleases which have been described, such as the one that occursnaturally in Tetrahymena thermophile, can be useful. See, for example,U.S. Pat. Nos. 4,987,071 and 6,423,885.

PTGS, e.g., RNAi, can also be used to inhibit the expression of a gene.For example, a construct can be prepared that includes a sequence thatis transcribed into an RNA that can anneal to itself, e.g., a doublestranded RNA having a stem-loop structure. In some embodiments, onestrand of the stem portion of a double stranded RNA comprises a sequencethat is similar or identical to the sense coding sequence or a fragmentthereof of an aluminum tolerance-modulating polypeptide, and that isfrom about 10 nucleotides to about 2,500 nucleotides in length. Thelength of the sequence that is similar or identical to the sense codingsequence can be from 10 nucleotides to 500 nucleotides, from 15nucleotides to 300 nucleotides, from 20 nucleotides to 100 nucleotides,or from 25 nucleotides to 100 nucleotides. The other strand of the stemportion of a double stranded RNA comprises a sequence that is similar oridentical to the antisense strand or a fragment thereof of the codingsequence of the aluminum tolerance-modulating polypeptide, and can havea length that is shorter, the same as, or longer than the correspondinglength of the sense sequence. In some cases, one strand of the stemportion of a double stranded RNA comprises a sequence that is similar oridentical to the 3′ or 5′ untranslated region, or a fragment thereof, ofan mRNA encoding an aluminum tolerance-modulating polypeptide, and theother strand of the stem portion of the double stranded RNA comprises asequence that is similar or identical to the sequence that iscomplementary to the 3′ or 5′ untranslated region, respectively, or afragment thereof, of the mRNA encoding the aluminum tolerance-modulatingpolypeptide. In other embodiments, one strand of the stem portion of adouble stranded RNA comprises a sequence that is similar or identical tothe sequence of an intron, or a fragment thereof, in the pre-mRNAencoding an aluminum tolerance-modulating polypeptide, and the otherstrand of the stem portion comprises a sequence that is similar oridentical to the sequence that is complementary to the sequence of theintron, or a fragment thereof, in the pre-mRNA.

The loop portion of a double stranded RNA can be from 3 nucleotides to5,000 nucleotides, e.g., from 3 nucleotides to 25 nucleotides, from 15nucleotides to 1,000 nucleotides, from 20 nucleotides to 500nucleotides, or from 25 nucleotides to 200 nucleotides. The loop portionof the RNA can include an intron or a fragment thereof. A doublestranded RNA can have zero, one, two, three, four, five, six, seven,eight, nine, ten, or more stem-loop structures.

A construct including a sequence that is operably linked to a regulatoryregion and a transcription termination sequence, and that is transcribedinto an RNA that can form a double stranded RNA, is transformed intoplants as described herein. Methods for using RNAi to inhibit theexpression of a gene are known to those of skill in the art. See, e.g.,U.S. Pat. Nos. 5,034,323; 6,326,527; 6,452,067; 6,573,099; 6,753,139;and 6,777,588. See also WO 97/01952; WO 98/53083; WO 99/32619; WO98/36083; and U.S. Patent Publications 20030175965, 20030175783,20040214330, and 20030180945.

Constructs containing regulatory regions operably linked to nucleic acidmolecules in sense orientation can also be used to inhibit theexpression of a gene. The transcription product can be similar oridentical to the sense coding sequence, or a fragment thereof, of analuminum tolerance-modulating polypeptide. The transcription productalso can be unpolyadenylated, lack a 5′ cap structure, or contain anunspliceable intron. Methods of inhibiting gene expression using afull-length cDNA as well as a partial cDNA sequence are known in theart. See, e.g., U.S. Pat. No. 5,231,020.

In some embodiments, a construct containing a nucleic acid having atleast one strand that is a template for both sense and antisensesequences that are complementary to each other is used to inhibit theexpression of a gene. The sense and antisense sequences can be part of alarger nucleic acid molecule or can be part of separate nucleic acidmolecules having sequences that are not complementary. The sense orantisense sequence can be a sequence that is identical or complementaryto the sequence of an mRNA, the 3′ or 5′ untranslated region of an mRNA,or an intron in a pre-mRNA encoding an aluminum tolerance-modulatingpolypeptide, or a fragment of such sequences. In some embodiments, thesense or antisense sequence is identical or complementary to a sequenceof the regulatory region that drives transcription of the gene encodingan aluminum tolerance-modulating polypeptide. In each case, the sensesequence is the sequence that is complementary to the antisensesequence.

The sense and antisense sequences can be a length greater than about 10nucleotides (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, or more nucleotides). For example, an antisensesequence can be 21 or 22 nucleotides in length. Typically, the sense andantisense sequences range in length from about 15 nucleotides to about30 nucleotides, e.g., from about 18 nucleotides to about 28 nucleotides,or from about 21 nucleotides to about 25 nucleotides.

In some embodiments, an antisense sequence is a sequence complementaryto an mRNA sequence, or a fragment thereof, encoding an aluminumtolerance-modulating polypeptide described herein. The sense sequencecomplementary to the antisense sequence can be a sequence present withinthe mRNA of the aluminum tolerance-modulating polypeptide. Typically,sense and antisense sequences are designed to correspond to a 15-30nucleotide sequence of a target mRNA such that the level of that targetmRNA is reduced.

In some embodiments, a construct containing a nucleic acid having atleast one strand that is a template for more than one sense sequence(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sense sequences) can be usedto inhibit the expression of a gene. Likewise, a construct containing anucleic acid having at least one strand that is a template for more thanone antisense sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreantisense sequences) can be used to inhibit the expression of a gene.For example, a construct can contain a nucleic acid having at least onestrand that is a template for two sense sequences and two antisensesequences. The multiple sense sequences can be identical or different,and the multiple antisense sequences can be identical or different. Forexample, a construct can have a nucleic acid having one strand that is atemplate for two identical sense sequences and two identical antisensesequences that are complementary to the two identical sense sequences.Alternatively, an isolated nucleic acid can have one strand that is atemplate for (1) two identical sense sequences 20 nucleotides in length,(2) one antisense sequence that is complementary to the two identicalsense sequences 20 nucleotides in length, (3) a sense sequence 30nucleotides in length, and (4) three identical antisense sequences thatare complementary to the sense sequence 30 nucleotides in length. Theconstructs provided herein can be designed to have a suitablearrangement of sense and antisense sequences. For example, two identicalsense sequences can be followed by two identical antisense sequences orcan be positioned between two identical antisense sequences.

A nucleic acid having at least one strand that is a template for one ormore sense and/or antisense sequences can be operably linked to aregulatory region to drive transcription of an RNA molecule containingthe sense and/or antisense sequence(s). In addition, such a nucleic acidcan be operably linked to a transcription terminator sequence, such asthe terminator of the nopaline synthase (nos) gene. In some cases, tworegulatory regions can direct transcription of two transcripts: one fromthe top strand, and one from the bottom strand. See, for example, Yan etal., Plant Physiol., 141:1508-1518 (2006). The two regulatory regionscan be the same or different. The two transcripts can formdouble-stranded RNA molecules that induce degradation of the target RNA.In some cases, a nucleic acid can be positioned within a T-DNA orplant-derived transfer DNA (P-DNA) such that the left and right T-DNAborder sequences or the left and right border-like sequences of theP-DNA flank, or are on either side of, the nucleic acid. See, U.S.Patent Publication No. 2006/0265788. The nucleic acid sequence betweenthe two regulatory regions can be from about 15 to about 300 nucleotidesin length. In some embodiments, the nucleic acid sequence between thetwo regulatory regions is from about 15 to about 200 nucleotides inlength, from about 15 to about 100 nucleotides in length, from about 15to about 50 nucleotides in length, from about 18 to about 50 nucleotidesin length, from about 18 to about 40 nucleotides in length, from about18 to about 30 nucleotides in length, or from about 18 to about 25nucleotides in length.

In some nucleic-acid based methods for inhibition of gene expression inplants, a suitable nucleic acid can be a nucleic acid analog. Nucleicacid analogs can be modified at the base moiety, sugar moiety, orphosphate backbone to improve, for example, stability, hybridization, orsolubility of the nucleic acid. Modifications at the base moiety includedeoxyuridine for deoxythymidine, and 5-methyl-2′-deoxycytidine and5-bromo-2′-deoxycytidine for deoxycytidine. Modifications of the sugarmoiety include modification of the 2′ hydroxyl of the ribose sugar toform 2′-O-methyl or 2′-O-allyl sugars. The deoxyribose phosphatebackbone can be modified to produce morpholino nucleic acids, in whicheach base moiety is linked to a six-membered morpholino ring, or peptidenucleic acids, in which the deoxyphosphate backbone is replaced by apseudopeptide backbone and the four bases are retained. See, forexample, Summerton and Weller, Antisense Nucleic Acid Drug Dev.,7:187-195 (1997); Hyrup et al., Bioorgan. Med. Chem., 4:5-23 (1996). Inaddition, the deoxyphosphate backbone can be replaced with, for example,a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite,or an alkyl phosphotriester backbone.

C. Constructs/Vectors

Recombinant constructs provided herein can be used to transform plantsor plant cells in order to modulate aluminum tolerance levels. Arecombinant nucleic acid construct can comprise a nucleic acid encodingan aluminum tolerance-modulating polypeptide as described herein,operably linked to a regulatory region suitable for expressing thealuminum tolerance-modulating polypeptide in the plant or cell. Thus, anucleic acid can comprise a coding sequence that encodes an aluminumtolerance-modulating polypeptide as set forth in SEQ ID NOs: 2, 3, 4, 5,7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 19, 20, 22, 24, 26, 28, 29, 31, 32,33, 35, 36, 38, 40, 41, 42, 43, 44, 45, 47, 48, 49, 50, 52, 54, 55, 56,57, 58, 59, 61, 63, 64, 65, 66, 67, 69, 70, 72, 73, 75, 77, 78, 79, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 93, 94, 96, 97, 98, 99, 100,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 113, 115, 117, 119,120, 121, 122, 124, 125, 126, 127, 129, 131, 132, 134, 136, 137, 138,140, 142, 144, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,158, 159, 160, 162, 163, 164, 166, 167, 169, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 190,191, 192, 193, 194, 195, 197, 198, 199, 200, 201, 202, 203, 205, 207,209, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 223, 224,225, 227, 228, 229, 230, 231, 232, 233, 234, 235, 237, 239, 241, 243,245, 246, 248, 250, 251, 253, 255, 257, 258, 259, 261, 263, 265, 267,269, 271, 273, 274, 276, 278, 280, 281, 283, 284, 285, 287, 288, 290,292, 293, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316,318, 320, 322, 324, 326, 328, 329, 331, 333, 335, 337, 338, 339, 341,342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 353, 355, 357, 358,360, 362, 364, 366, 368, 369, 371, 373, 375, 376, 377, 378, 379, 380,382, 384, 386, 388, 390, 392, 393, 395, 396, 397, 398, 399, 400, 401,402, 404, 405, 407, 409, 411, 413, 414, 416, 418, 420, 421, 423, 424,425, 426, 428, 429, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,441, 442, 443, 444, 445, 446, 447, 449, 451, 453, 454, 456, 457, 458,459, 460, 461, 462, 463, 465, 467, 468, 469, 470, 471, 472, 473, 474,475, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500,502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528,530, 532, 534, 535, 537, 539, 540, 542, 544, 545, 547, 549, 551, 553,555, 557, 558, 560, 562, 564, 568, 570, 572, 574, 576, 578, 579, 580,581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607,608, 610, 612, 614, 616, 618, 620, 622, 624, 625, 627, 629, 631, 633,635, 637, 639, 641, 643, 645, 647, 649, 650, 651, 652, 653, 654, 655,656, 657, 658, 660, 662, 664, 666, 668, 669, 670, 671, 672, 673, 674,675, 676, 677, 679, 681, 683, 685, 686, 687, 689, 691, 693, 695, 696,698, 699, 701, 702, 703, 704, 705, 706, 707, 709, 710, 711, 712, 713,714, 715, 716, 717, 718, 719, 721, 722, 723, 724, 725, 726, 727, 728,729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 742, 744,745, 746, 747, 749, 750, 752, 753, 754, 755, 757, 759, 760, 761, 762,764, 765, 766, 767, 768, 769, 770, 771, and 772. Examples of nucleicacids encoding aluminum tolerance-modulating polypeptides are set forthin SEQ ID NOs: 1, 6, 16, 18, 21, 23, 25, 27, 30, 34, 37, 39, 46, 51, 53,60, 62, 68, 71, 74, 76, 80, 92, 95, 101, 112, 114, 116, 118, 123, 128,130, 133, 135, 139, 141, 143, 145, 157, 161, 165, 168, 170, 189, 196,204, 206, 208, 210, 222, 226, 236, 238, 240, 242, 244, 247, 249, 252,254, 256, 260, 262, 264, 266, 268, 270, 272, 275, 277, 279, 282, 286,289, 291, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317,319, 321, 323, 325, 327, 330, 332, 334, 336, 340, 352, 354, 356, 359,361, 363, 365, 367, 370, 372, 374, 381, 383, 385, 387, 389, 391, 394,403, 406, 408, 410, 412, 415, 417, 419, 422, 427, 430, 448, 450, 452,455, 464, 466, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497,499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525,527, 529, 531, 533, 536, 538, 541, 543, 546, 548, 550, 552, 554, 556,559, 561, 563, 567, 569, 571, 573, 575, 577, 582, 584, 586, 588, 590,592, 594, 596, 598, 600, 602, 604, 606, 609, 611, 613, 615, 617, 619,621, 623, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648,659, 661, 663, 665, 667, 678, 680, 682, 684, 688, 690, 692, 694, 697,700, 708, 720, 741, 743, 748, 751, 756, 758, and 763, or in the SequenceListing. The aluminum tolerance-modulating polypeptide encoded by arecombinant nucleic acid can be a native aluminum tolerance-modulatingpolypeptide, or can be heterologous to the cell. In some cases, therecombinant construct contains a nucleic acid that inhibits expressionof an aluminum tolerance-modulating polypeptide, operably linked to aregulatory region. Examples of suitable regulatory regions are describedin the section entitled “Regulatory Regions.”

Vectors containing recombinant nucleic acid constructs such as thosedescribed herein also are provided. Suitable vector backbones include,for example, those routinely used in the art such as plasmids, viruses,artificial chromosomes, BACs, YACs, or PACs. Suitable expression vectorsinclude, without limitation, plasmids and viral vectors derived from,for example, bacteriophage, baculoviruses, and retroviruses. Numerousvectors and expression systems are commercially available from suchcorporations as Novagen® (Madison, Wis.), Clontech® (Palo Alto, Calif.),Stratagene® (La Jolla, Calif.), and Invitrogen/Life Technologies®(Carlsbad, Calif.).

The vectors provided herein also can include, for example, origins ofreplication, selectable phenotype on a plant cell. For example, a markercan confer biocide scaffold attachment regions (SARs), and/or markers. Amarker gene can confer a resistance, such as resistance to an antibiotic(e.g., kanamycin, G418, bleomycin, or hygromycin), or an herbicide(e.g., glyphosate, chlorsulfuron or phosphinothricin). In addition, anexpression vector can include a tag sequence designed to facilitatemanipulation or detection (e.g., purification or localization) of theexpressed polypeptide. Tag sequences, such as luciferase,β-glucuronidase (GUS), green fluorescent protein (GFP), glutathioneS-transferase (GST), polyhistidine, c-myc, hemagglutinin, or Flag™ tag(Kodak, New Haven, Conn.) sequences typically are expressed as a fusionwith the encoded polypeptide. Such tags can be inserted anywhere withinthe polypeptide, including at either the carboxyl or amino terminus.

D. Regulatory Regions

The choice of regulatory regions to be included in a recombinantconstruct depends upon several factors, including, but not limited to,efficiency, selectability, inducibility, desired expression level, andcell- or tissue-preferential expression. It is a routine matter for oneof skill in the art to modulate the expression of a coding sequence byappropriately selecting and positioning regulatory regions relative tothe coding sequence. Transcription of a nucleic acid can be modulated ina similar manner.

Some suitable regulatory regions initiate transcription only, orpredominantly, in certain cell types. Methods for identifying andcharacterizing regulatory regions in plant genomic DNA are known,including, for example, those described in the following references:Jordano et al., Plant Cell, 1:855-866 (1989); Bustos et al., Plant Cell,1:839-854 (1989); Green et al., EMBO J., 7:4035-4044 (1988); Meier etal., Plant Cell, 3:309-316 (1991); and Zhang et al., Plant Physiology,110:1069-1079 (1996).

Examples of various classes of regulatory regions are described below.Some of the regulatory regions indicated below as well as additionalregulatory regions are described in more detail in U.S. PatentApplication Ser. Nos. 60/505,689; 60/518,075; 60/544,771; 60/558,869;60/583,691; 60/619,181; 60/637,140; 60/757,544; 60/776,307; 10/957,569;11/058,689; 11/172,703; 11/208,308; 11/274,890; 60/583,609; 60/612,891;11/097,589; 11/233,726; 11/408,791; 11/414,142; 10/950,321; 11/360,017;PCT/US05/011105; PCT/US05/23639; PCT/US05/034308; PCT/US05/034343; andPCT/US06/038236; PCT/US06/040572; PCT/US07/62762; PCT/US2009/032485; andPCT/US2009/038792.

For example, the sequences of regulatory regions p326, YP0144, YP0190,p13879, YP0050, p32449, 21876, YP0158, YP0214, YP0380, PT0848, PT0633,YP0128, YP0275, PT0660, PT0683, PT0758, PT0613, PT0672, PT0688, PT0837,YP0092, PT0676, PT0708, YP0396, YP0007, YP0111, YP0103, YP0028, YP0121,YP0008, YP0039, YP0115, YP0119, YP0120, YP0374, YP0101, YP0102, YP0110,YP0117, YP0137, YP0285, YP0212, YP0097, YP0107, YP0088, YP0143, YP0156,PT0650, PT0695, PT0723, PT0838, PT0879, PT0740, PT0535, PT0668, PT0886,PT0585, YP0381, YP0337, PT0710, YP0356, YP0385, YP0384, YP0286, YP0377,PD1367, PT0863, PT0829, PT0665, PT0678, YP0086, YP0188, YP0263, PT0743and YP0096 are set forth in the sequence listing of PCT/US06/040572; thesequence of regulatory region PT0625 is set forth in the sequencelisting of PCT/US05/034343; the sequences of regulatory regions PT0623,YP0388, YP0087, YP0093, YP0108, YP0022 and YP0080 are set forth in thesequence listing of U.S. patent application Ser. No. 11/172,703; thesequence of regulatory region PRO924 is set forth in the sequencelisting of PCT/US07/62762; and the sequences of regulatory regionsp530c10, pOsFIE2-2, pOsMEA, pOsYp102, and pOsYp285 are set forth in thesequence listing of PCT/US06/038236.

It will be appreciated that a regulatory region may meet criteria forone classification based on its activity in one plant species, and yetmeet criteria for a different classification based on its activity inanother plant species.

i. Broadly Expressing Promoters

A promoter can be said to be “broadly expressing” when it promotestranscription in many, but not necessarily all, plant tissues. Forexample, a broadly expressing promoter can promote transcription of anoperably linked sequence in one or more of the shoot, shoot tip (apex),and leaves, but weakly or not at all in tissues such as roots or stems.As another example, a broadly expressing promoter can promotetranscription of an operably linked sequence in one or more of the stem,shoot, shoot tip (apex), and leaves, but can promote transcriptionweakly or not at all in tissues such as reproductive tissues of flowersand developing seeds. Non-limiting examples of broadly expressingpromoters that can be included in the nucleic acid constructs providedherein include the p326, YP0144, YP0190, p13879, YP0050, p32449, 21876,YP0158, YP0214, YP0380, PT0848, and PT0633 promoters. Additionalexamples include the cauliflower mosaic virus (CaMV) ³⁵S promoter, themannopine synthase (MAS) promoter, the l′ or 2′ promoters derived fromT-DNA of Agrobacterium tumefaciens, the figwort mosaic virus 34Spromoter, actin promoters such as the rice actin promoter, and ubiquitinpromoters such as the maize ubiquitin-1 promoter. In some cases, theCaMV ³⁵S promoter is excluded from the category of broadly expressingpromoters.

Another example of a broad promoter is the sequence of regulatory regionPD3141 set forth in the sequence listing of PCT/US2009/032485. Therein,the expression pattern of the PD3141 regulatory region is described forTO rice plants overexpressing a construct comprising PD3141 drivingexpression of EGFP. In seedlings, expression was observed in: Tiller:not-specific; Main culm: not-specific; Root: not-specific; Leaf:not-specific; and Meristem: not-specific. In mature plants, expressionwas observed in: Main culm: bundle sheath, endodermis, epidermis,internode, ligule, node, pericycle, phloem, sclerenchyma layer,vasculature, xylem; Root: cortex, vascular; Panicle: flag leaf, ovary,peduncle, primary branch, rachilla, rachis, spiklet; Spiklet: flag leaf,floret(palea), lemma, ovule, pedicle, pollen, seed, stigma; Leaf:epidermis, leaf blade, leaf sheath, mesophyll; and Meristem: floralmeristem, shoot apical meristem, vegetative meristem.

Another example of a broad promoter is the sequence of regulatory regionp326 set forth in the sequence listing of U.S. application Ser. No.10/981,334. Therein, the expression pattern of the p326 regulatoryregion is described for Arabidopsis plants. p326 expressed throughoutmost mature tissues screened. Expression was somewhat higher inepidermal, vascular and photosynthetic tissue of seedling. Linescharacterized went through several generations.

Another example of a broad promoter is the sequence of regulatory regionPD2995 (a 600 bp version of p326) set forth in the sequence listing ofPCT/US2009/32485. In TO rice plants, PD2995 expresses very weaklythroughout all tissues of the plant in both seedling and mature stages.Strongest expression detected in root tissue and embryo.

ii. Root Promoters

Root-active promoters confer transcription in root tissue, e.g., rootendodermis, root epidermis, or root vascular tissues. In someembodiments, root-active promoters are root-preferential promoters,i.e., confer transcription only or predominantly in root tissue.Root-preferential promoters include the YP0128, YP0275, PT0625, PT0660,PT0683, and PT0758 promoters. Other root-preferential promoters includethe PT0613, PT0672, PT0688, and PT0837 promoters, which drivetranscription primarily in root tissue and to a lesser extent in ovulesand/or seeds. Other examples of root-preferential promoters include theroot-specific subdomains of the CaMV ³⁵S promoter (Lam et al., Proc.Natl. Acad. Sci. USA, 86:7890-7894 (1989)), root cell specific promotersreported by Conkling et al., Plant Physiol., 93:1203-1211 (1990), andthe tobacco RD2 promoter.

Another example of a root promoter is the sequence of regulatory regionPD3561 set forth in the sequence listing of PCT/US2009/038792. Therein,the expression pattern of the PD3561 regulatory region is described forTO rice plants overexpressing a construct comprising PD3561 drivingexpression of EGFP. Expression was observed in roots of seedlings in thecortex, epidermis, and vascular tissues. In mature plants, expressionwas observed strongly throughout the root with the exception of the rootcap and in the cortex, epidermis, and vascular tissues.

iii. Maturing Endosperm Promoters

In some embodiments, promoters that drive transcription in maturingendosperm can be useful. Transcription from a maturing endospermpromoter typically begins after fertilization and occurs primarily inendosperm tissue during seed development and is typically highest duringthe cellularization phase. Most suitable are promoters that are activepredominantly in maturing endosperm, although promoters that are alsoactive in other tissues can sometimes be used. Non-limiting examples ofmaturing endosperm promoters that can be included in the nucleic acidconstructs provided herein include the napin promoter, the Arcelin-5promoter, the phaseolin promoter (Bustos et al., Plant Cell,1(9):839-853 (1989)), the soybean trypsin inhibitor promoter (Riggs etal., Plant Cell, 1(6):609-621 (1989)), the ACP promoter (Baerson et al.,Plant Mol. Biol., 22(2):255-267 (1993)), the stearoyl-ACP desaturasepromoter (Slocombe et al., Plant Physiol., 104(4):167-176 (1994)), thesoybean a′ subunit of β-conglycinin promoter (Chen et al., Proc. Natl.Acad. Sci. USA, 83:8560-8564 (1986)), the oleosin promoter (Hong et al.,Plant Mol. Biol., 34(3):549-555 (1997)), and zein promoters, such as the15 kD zein promoter, the 16 kD zein promoter, 19 kD zein promoter, 22 kDzein promoter and 27 kD zein promoter. Also suitable are the Osgt-1promoter from the rice glutelin-1 gene (Zheng et al., Mol. Cell. Biol.,13:5829-5842 (1993)), the beta-amylase promoter, and the barley hordeinpromoter. Other maturing endosperm promoters include the YP0092, PT0676,and PT0708 promoters.

iv. Ovary Tissue Promoters

Promoters that are active in ovary tissues such as the ovule wall andmesocarp can also be useful, e.g., a polygalacturonidase promoter, thebanana TRX promoter, the melon actin promoter, YP0396, and PT0623.Examples of promoters that are active primarily in ovules includeYP0007, YP0111, YP0092, YP0103, YP0028, YP0121, YP0008, YP0039, YP0115,YP0119, YP0120, and YP0374.

v. Embryo Sac/Early Endosperm Promoters

To achieve expression in embryo sac/early endosperm, regulatory regionscan be used that are active in polar nuclei and/or the central cell, orin precursors to polar nuclei, but not in egg cells or precursors to eggcells. Most suitable are promoters that drive expression only orpredominantly in polar nuclei or precursors thereto and/or the centralcell. A pattern of transcription that extends from polar nuclei intoearly endosperm development can also be found with embryo sac/earlyendosperm-preferential promoters, although transcription typicallydecreases significantly in later endosperm development during and afterthe cellularization phase. Expression in the zygote or developing embryotypically is not present with embryo sac/early endosperm promoters.

Promoters that may be suitable include those derived from the followinggenes: Arabidopsis viviparous-1 (see, GenBank No. U93215); Arabidopsisatmycl (see, Urao, Plant Mol. Biol., 32:571-57 (1996); Conceicao, Plant,5:493-505 (1994)); Arabidopsis FIE (GenBank No. AF129516); ArabidopsisMEA; Arabidopsis FIS2 (GenBank No. AF096096); and FIE 1.1 (U.S. Pat. No.6,906,244). Other promoters that may be suitable include those derivedfrom the following genes: maize MAC1 (see, Sheridan, Genetics,142:1009-1020 (1996)); maize Cat3 (see, GenBank No. L05934; Abler, PlantMol. Biol., 22:10131-1038 (1993)). Other promoters include the followingArabidopsis promoters: YP0039, YP0101, YP0102, YP0110, YP0117, YP0119,YP0137, DME, YP0285, and YP0212. Other promoters that may be usefulinclude the following rice promoters: p530c10, pOsFIE2-2, pOsMEA,pOsYp102, and pOsYp285.

vi. Embryo Promoters

Regulatory regions that preferentially drive transcription in zygoticcells following fertilization can provide embryo-preferentialexpression. Most suitable are promoters that preferentially drivetranscription in early stage embryos prior to the heart stage, butexpression in late stage and maturing embryos is also suitable.Embryo-preferential promoters include the barley lipid transfer protein(Ltp1) promoter (Plant Cell Rep 20:647-654 (2001)), YP0097, YP0107,YP0088, YP0143, YP0156, PT0650, PT0695, PT0723, PT0838, PT0879, andPT0740.

vii. Photosynthetic Tissue Promoters

Promoters active in photosynthetic tissue confer transcription in greentissues such as leaves and stems. Most suitable are promoters that driveexpression only or predominantly in such tissues. Examples of suchpromoters include the ribulose-1,5-bisphosphate carboxylase (RbcS)promoters such as the RbcS promoter from eastern larch (Larix laricina),the pine cab6 promoter (Yamamoto et al., Plant Cell Physiol., 35:773-778(1994)), the Cab-1 promoter from wheat (Fejes et al., Plant Mol. Biol.,15:921-932 (1990)), the CAB-1 promoter from spinach (Lubberstedt et al.,Plant Physiol., 104:997-1006 (1994)), the cab1R promoter from rice (Luanet al., Plant Cell, 4:971-981 (1992)), the pyruvate orthophosphatedikinase (PPDK) promoter from corn (Matsuoka et al., Proc. Natl. Acad.Sci. USA, 90:9586-9590 (1993)), the tobacco Lhcb1*2 promoter (Cerdan etal., Plant Mol. Biol., 33:245-255 (1997)), the Arabidopsis thaliana SUC2sucrose-H+ symporter promoter (Truernit et al., Planta, 196:564-570(1995)), and thylakoid membrane protein promoters from spinach (psaD,psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS). Other photosynthetic tissuepromoters include PT0535, PT0668, PT0886, YP0144, YP0380 and PT0585.

viii. Vascular Tissue Promoters

Examples of promoters that have high or preferential activity invascular bundles include YP0087, YP0093, YP0108, YP0022, and YP0080.Other vascular tissue-preferential promoters include the glycine-richcell wall protein GRP 1.8 promoter (Keller and Baumgartner, Plant Cell,3(10):1051-1061 (1991)), the Commelina yellow mottle virus (CoYMV)promoter (Medberry et al., Plant Cell, 4(2):185-192 (1992)), and therice tungro bacilliform virus (RTBV) promoter (Dai et al., Proc. Natl.Acad. Sci. USA, 101(2):687-692 (2004)).

ix. Inducible Promoters

Inducible promoters confer transcription in response to external stimulisuch as chemical agents or environmental stimuli. For example, induciblepromoters can confer transcription in response to hormones such asgiberellic acid or ethylene, or in response to light or drought.Examples of drought-inducible promoters include YP0380, PT0848, YP0381,YP0337, PT0633, YP0374, PT0710, YP0356, YP0385, YP0396, YP0388, YP0384,PT0688, YP0286, YP0377, PD1367, and PD0901. Examples ofnitrogen-inducible promoters include PT0863, PT0829, PT0665, and PT0886.Examples of shade-inducible promoters include PRO924 and PT0678. Anexample of a promoter induced by salt is rd29A (Kasuga et al. (1999)Nature Biotech 17: 287-291).

x. Basal Promoters

A basal promoter is the minimal sequence necessary for assembly of atranscription complex required for transcription initiation. Basalpromoters frequently include a “TATA box” element that may be locatedbetween about 15 and about 35 nucleotides upstream from the site oftranscription initiation. Basal promoters also may include a “CCAAT box”element (typically the sequence CCAAT) and/or a GGGCG sequence, whichcan be located between about 40 and about 200 nucleotides, typicallyabout 60 to about 120 nucleotides, upstream from the transcription startsite.

xi. Stem Promoters

A stem promoter may be specific to one or more stem tissues or specificto stem and other plant parts. Stem promoters may have high orpreferential activity in, for example, epidermis and cortex, vascularcambium, procambium, or xylem. Examples of stem promoters include YP0018which is disclosed in US20060015970 and CryIA(b) and CryIA(c) (Braga etal. 2003, Journal of New Seeds 5:209-221).

xii. Other Promoters

Other classes of promoters include, but are not limited to,shoot-preferential, callus-preferential, trichome cell-preferential,guard cell-preferential such as PT0678, tuber-preferential, parenchymacell-preferential, and senescence-preferential promoters. Promotersdesignated YP0086, YP0188, YP0263, PT0758, PT0743, PT0829, YP0119, andYP0096, as described in the above-referenced patent applications, mayalso be useful.

xiii. Other Regulatory Regions

A 5′ untranslated region (UTR) can be included in nucleic acidconstructs described herein. A 5′ UTR is transcribed, but is nottranslated, and lies between the start site of the transcript and thetranslation initiation codon and may include the +1 nucleotide. A 3′ UTRcan be positioned between the translation termination codon and the endof the transcript. UTRs can have particular functions such as increasingmRNA stability or attenuating translation. Examples of 3′ UTRs include,but are not limited to, polyadenylation signals and transcriptiontermination sequences, e.g., a nopaline synthase termination sequence.

It will be understood that more than one regulatory region may bepresent in a recombinant polynucleotide, e.g., introns, enhancers,upstream activation regions, transcription terminators, and inducibleelements. Thus, for example, more than one regulatory region can beoperably linked to the sequence of a polynucleotide encoding an aluminumtolerance-modulating polypeptide.

Regulatory regions, such as promoters for endogenous genes, can beobtained by chemical synthesis or by subcloning from a genomic DNA thatincludes such a regulatory region. A nucleic acid comprising such aregulatory region can also include flanking sequences that containrestriction enzyme sites that facilitate subsequent manipulation.

IV. Transgenic Plants and Plant Cells

A. Transformation

The invention also features transgenic plant cells and plants comprisingat least one recombinant nucleic acid construct described herein. Aplant or plant cell can be transformed by having a construct integratedinto its genome, i.e., can be stably transformed. Stably transformedcells typically retain the introduced nucleic acid with each celldivision. A plant or plant cell can also be transiently transformed suchthat the construct is not integrated into its genome. Transientlytransformed cells typically lose all or some portion of the introducednucleic acid construct with each cell division such that the introducednucleic acid cannot be detected in daughter cells after a sufficientnumber of cell divisions. Both transiently transformed and stablytransformed transgenic plants and plant cells can be useful in themethods described herein.

Transgenic plant cells used in methods described herein can constitutepart or all of a whole plant. Such plants can be grown in a mannersuitable for the species under consideration, either in a growthchamber, a greenhouse, or in a field. Transgenic plants can be bred asdesired for a particular purpose, e.g., to introduce a recombinantnucleic acid into other lines, to transfer a recombinant nucleic acid toother species, or for further selection of other desirable traits.Alternatively, transgenic plants can be propagated vegetatively forthose species amenable to such techniques. As used herein, a transgenicplant also refers to progeny of an initial transgenic plant provided theprogeny inherits the transgene. Seeds produced by a transgenic plant canbe grown and then selfed (or outcrossed and selfed) to obtain seedshomozygous for the nucleic acid construct.

Transgenic plants can be grown in suspension culture, or tissue or organculture. For the purposes of this invention, solid and/or liquid tissueculture techniques can be used. When using solid medium, transgenicplant cells can be placed directly onto the medium or can be placed ontoa filter that is then placed in contact with the medium. When usingliquid medium, transgenic plant cells can be placed onto a flotationdevice, e.g., a porous membrane that contacts the liquid medium. A solidmedium can be, for example, Murashige and Skoog (MS) medium containingagar and a suitable concentration of an auxin, e.g.,2,4-dichlorophenoxyacetic acid (2,4-D), and a suitable concentration ofa cytokinin, e.g., kinetin.

When transiently transformed plant cells are used, a reporter sequenceencoding a reporter polypeptide having a reporter activity can beincluded in the transformation procedure and an assay for reporteractivity or expression can be performed at a suitable time aftertransformation. A suitable time for conducting the assay typically isabout 1-21 days after transformation, e.g., about 1-14 days, about 1-7days, or about 1-3 days. The use of transient assays is particularlyconvenient for rapid analysis in different species, or to confirmexpression of a heterologous aluminum tolerance-modulating polypeptidewhose expression has not previously been confirmed in particularrecipient cells.

Techniques for introducing nucleic acids into monocotyledonous anddicotyledonous plants are known in the art, and include, withoutlimitation, Agrobacterium-mediated transformation, viral vector-mediatedtransformation, electroporation and particle gun transformation, e.g.,U.S. Pat. Nos. 5,538,880; 5,204,253; 6,329,571 and 6,013,863. If a cellor cultured tissue is used as the recipient tissue for transformation,plants can be regenerated from transformed cultures if desired, bytechniques known to those skilled in the art.

B. Screening/Selection

A population of transgenic plants can be screened and/or selected forthose members of the population that have a trait or phenotype conferredby expression of the transgene. For example, a population of progeny ofa single transformation event can be screened for those plants having adesired level of expression of an aluminum tolerance-modulatingpolypeptide or nucleic acid. In some embodiments, a population of plantscan be selected that has increased tolerance to elevated levels of Al³⁺in soil without increased tolerance to drought or elevated salinelevels. Plant species vary in their capacity to tolerate salinity.“Salinity” refers to a set of environmental conditions under which aplant will begin to suffer the effects of elevated salt concentration,such as ion imbalance, decreased stomatal conductance, decreasedphotosynthesis, decreased growth rate, increased cell death, loss ofturgor (wilting), or ovule abortion. For these reasons, plantsexperiencing salinity stress typically exhibit a significant reductionin biomass and/or yield.

Physical and biochemical methods can be used to identify expressionlevels. These include Southern analysis or PCR amplification fordetection of a polynucleotide; Northern blots, S1 RNase protection,primer-extension, or RT-PCR amplification for detecting RNA transcripts;enzymatic assays for detecting enzyme or ribozyme activity ofpolypeptides and polynucleotides; and protein gel electrophoresis,Western blots, immunoprecipitation, and enzyme-linked immunoassays todetect polypeptides. Other techniques such as in situ hybridization,enzyme staining, and immunostaining also can be used to detect thepresence or expression of polypeptides and/or polynucleotides. Methodsfor performing all of the referenced techniques are known. As analternative, a population of plants comprising independenttransformation events can be screened for those plants having a desiredtrait, such as increased tolerance to elevated levels of aluminum.Selection and/or screening can be carried out over one or moregenerations, and/or in more than one geographic location. In some cases,transgenic plants can be grown and selected under conditions whichinduce a desired phenotype or are otherwise necessary to produce adesired phenotype in a transgenic plant. In addition, selection and/orscreening can be applied during a particular developmental stage inwhich the phenotype is expected to be exhibited by the plant. Selectionand/or screening can be carried out to choose those transgenic plantshaving a statistically significant difference in an aluminum tolerancelevel relative to a control plant that lacks the transgene. Selected orscreened transgenic plants have an altered phenotype as compared to acorresponding control plant, as described in the “Transgenic PlantPhenotypes” section herein.

C. Plant Species

The polynucleotides and vectors described herein can be used totransform a number of monocotyledonous and dicotyledonous plants andplant cell systems, including species from one of the followingfamilies: Acanthaceae, Alliaceae, Alstroemeriaceae, Amaryllidaceae,Apocynaceae, Arecaceae, Asteraceae, Berberidaceae, Bixaceae,Brassicaceae, Bromeliaceae, Cannabaceae, Caryophyllaceae,Cephalotaxaceae, Chenopodiaceae, Colchicaceae, Cucurbitaceae,Dioscoreaceae, Ephedraceae, Erythroxylaceae, Euphorbiaceae, Fabaceae,Lamiaceae, Linaceae, Lycopodiaceae, Malvaceae, Melanthiaceae, Musaceae,Myrtaceae, Nyssaceae, Papaveraceae, Pinaceae, Plantaginaceae, Poaceae,Rosaceae, Rubiaceae, Salicaceae, Sapindaceae, Solanaceae, Taxaceae,Theaceae, or Vitaceae.

Suitable species may include members of the genus Abelmoschus, Abies,Acer, Agrostis, Allium, Alstroemeria, Ananas, Andrographis, Andropogon,Artemisia, Arundo, Atropa, Berberis, Beta, Bixa, Brassica, Calendula,Camellia, Camptotheca, Cannabis, Capsicum, Carthamus, Catharanthus,Cephalotaxus, Chrysanthemum, Cinchona, Citrullus, Coffea, Colchicum,Coleus, Cucumis, Cucurbita, Cynodon, Datura, Dianthus, Digitalis,Dioscorea, Elaeis, Ephedra, Erianthus, Erythroxylum, Eucalyptus,Festuca, Fragaria, Galanthus, Glycine, Gossypium, Helianthus, Hevea,Hordeum, Hyoscyamus, Jatropha, Lactuca, Linum, Lolium, Lupinus,Lycopersicon, Lycopodium, Manihot, Medicago, Mentha, Miscanthus, Musa,Nicotiana, Oryza, Panicum, Papaver, Parthenium, Pennisetum, Petunia,Phalaris, Phleum, Pinus, Poa, Poinsettia, Populus, Rauwolfia, Ricinus,Rosa, Saccharum, Salix, Sanguinaria, Scopolia, Secale, Solanum, Sorghum,Spartina, Spinacea, Tanacetum, Taxus, Theobroma, Triticosecale,Triticum, Uniola, Veratrum, Vinca, Vitis, and Zea.

Suitable species include Panicum spp., Sorghum spp., Miscanthus spp.,Saccharum spp., Erianthus spp., Populus spp., Andropogon gerardii (bigbluestem), Pennisetum purpureum (elephant grass), Phalaris arundinacea(reed canarygrass), Cynodon dactylon (bermudagrass), Festuca arundinacea(tall fescue), Spartina pectinata (prairie cord-grass), Medicago sativa(alfalfa), Arundo donax (giant reed), Secale cereale (rye), Salix spp.(willow), Eucalyptus spp. (eucalyptus), Triticosecale (triticum—wheat Xrye) and bamboo.

Suitable species also include Helianthus annuus (sunflower), Carthamustinctorius (safflower), Jatropha curcas (jatropha), Ricinus communis(castor), Elaeis guineensis (palm), Linum usitatissimum (flax), andBrassica juncea.

Suitable species also include Beta vulgaris (sugarbeet), and Manihotesculenta (cassava)

Suitable species also include Lycopersicon esculentum (tomato), Lactucasativa (lettuce), Musa paradisiaca (banana), Solanum tuberosum (potato),Brassica oleracea (broccoli, cauliflower, Brussels sprouts), Camelliasinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa),Coffea arabica (coffee), Vitis vinifera (grape), Ananas comosus(pineapple), Capsicum annum (hot & sweet pepper), Allium cepa (onion),Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima(squash), Cucurbita moschata (squash), Spinacea oleracea (spinach),Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), andSolanum melongena (eggplant).

Suitable species also include Papaver somniferum (opium poppy), Papaverorientale, Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabissativa, Camptotheca acuminate, Catharanthus roseus, Vinca rosea,Cinchona officinalis, Colchicum autumnale, Veratrum californica,Digitalis lanata, Digitalis purpurea, Dioscorea spp., Andrographispaniculata, Atropa belladonna, Datura stomonium, Berberis spp.,Cephalotaxus spp., Ephedra sinica, Ephedra spp., Erythroxylum coca,Galanthus wornorii, Scopolia spp., Lycopodium serratum (Huperziaserrata), Lycopodium spp., Rauwolfia serpentina, Rauwolfia spp.,Sanguinaria canadensis, Hyoscyamus spp., Calendula officinalis,Chrysanthemum parthenium, Coleus forskohlii, and Tanacetum parthenium.

Suitable species also include Parthenium argentatum (guayule), Heveaspp. (rubber), Mentha spicata (mint), Mentha piperita (mint), Bixaorellana, and Alstroemeria spp.

Suitable species also include Rosa spp. (rose), Dianthus caryophyllus(carnation), Petunia spp. (petunia) and Poinsettia pulcherrima(poinsettia).

Suitable species also include Nicotiana tabacum (tobacco), Lupinus albus(lupin), Uniola paniculata (oats), bentgrass (Agrostis spp.), Populustremuloides (aspen), Pinus spp. (pine), Abies spp. (fir), Acer spp.(maple), Hordeum vulgare (barley), Poa pratensis (bluegrass), Loliumspp. (ryegrass) and Phleum pratense (timothy).

In some embodiments, a suitable species can be a wild, weedy, orcultivated Pennisetum species such as, but not limited to, Pennisetumalopecuroides, Pennisetum arnhemicum, Pennisetum caffrum, Pennisetumclandestinum, Pennisetum divisum, Pennisetum glaucum, Pennisetumlatifolium, Pennisetum macrostachyum, Pennisetum macrourum, Pennisetumorientate, Pennisetum pedicellatum, Pennisetum polystachion, Pennisetumpolystachion ssp. Setosum, Pennisetum purpureum, Pennisetum setaceum,Pennisetum subangustum, Pennisetum typhoides, Pennisetum villosum, orhybrids thereof (e.g., Pennisetum purpureum x Pennisetum typhoidum).

In some embodiments, a suitable species can be a wild, weedy, orcultivated Miscanthus species and/or variety such as, but not limitedto, Miscanthus x giganteus, Miscanthus sinensis, Miscanthus x ogiformis,Miscanthus floridulus, Miscanthus transmorrisonensis, Miscanthusoligostachyus, Miscanthus nepalensis, Miscanthus sacchariflorus,Miscanthus x giganteus ‘Amuri’, Miscanthus x giganteus ‘Nagara’,Miscanthus x giganteus ‘Illinois’, Miscanthus sinensis var. ‘Goliath’,Miscanthus sinensis var. ‘Roland’, Miscanthus sinensis var. ‘Africa’,Miscanthus sinensis var. ‘Fern Osten’, Miscanthus sinensis var.gracillimus, Miscanthus sinensis var. variegates, Miscanthus sinensisvar. purpurascens, Miscanthus sinensis var. ‘Malepartus’, Miscanthussacchariflorus var. ‘Robusta’, Miscanthus sinensis var. ‘Silberfedher’(aka. Silver Feather), Miscanthus transmorrisonensis, Miscanthuscondensatus, Miscanthus yakushimanum, Miscanthus var. ‘Alexander’,Miscanthus var. ‘Adagio’, Miscanthus var. ‘Autumn Light’, Miscanthusvar. ‘Cabaret’, Miscanthus var. ‘Condensatus’, Miscanthus var.‘Cosmopolitan’, Miscanthus var. ‘Dixieland’, Miscanthus var. ‘GildedTower’ (U.S. Pat. No. PP14,743), Miscanthus var. ‘Gold Bar’ (U.S. Pat.No. PP15,193), Miscanthus var. ‘Gracillimus’, Miscanthus var.‘Graziella’, Miscanthus var. ‘Grosse Fontaine’, Miscanthus var. ‘Hinjoaka Little Nicky’™, Miscanthus var. ‘Juli’, Miscanthus var. ‘Kaskade’,Miscanthus var. ‘Kirk Alexander’, Miscanthus var. ‘Kleine Fontaine’,Miscanthus var. ‘Kleine Silberspinne’ (aka. ‘Little Silver Spider’),Miscanthus var. ‘Little Kitten’, Miscanthus var. ‘Little Zebra’ (U.S.Pat. No. PP13,008), Miscanthus var. ‘Lottum’, Miscanthus var.‘Malepartus’, Miscanthus var. ‘Morning Light’, Miscanthus var.‘Mysterious Maiden’ (U.S. Pat. No. PP16,176), Miscanthus var. ‘Nippon’,Miscanthus var. ‘November Sunset’, Miscanthus var. ‘Parachute’,Miscanthus var. ‘Positano’, Miscanthus var. ‘Puenktchen’(aka ‘LittleDot’), Miscanthus var. ‘Rigoletto’, Miscanthus var. ‘Sarabande’,Miscanthus var. ‘Silberpfeil’ (aka. Silver Arrow), Miscanthus var.‘Silverstripe’, Miscanthus var. ‘Super Stripe’ (U.S. Pat. No. PP18,161),Miscanthus var. ‘Strictus’, or Miscanthus var. ‘Zebrinus’.

In some embodiments, a suitable species can be a wild, weedy, orcultivated sorghum species and/or variety such as, but not limited to,Sorghum almum, Sorghum amplum, Sorghum angustum, Sorghum arundinaceum,Sorghum bicolor (such as bicolor, guinea, caudatum, kafir, and durra),Sorghum brachypodum, Sorghum bulbosum, Sorghum burmahicum, Sorghumcontroversum, Sorghum drummondii, Sorghum ecarinatum, Sorghum exstans,Sorghum grande, Sorghum halepense, Sorghum interjectum, Sorghum intrans,Sorghum laxiflorum, Sorghum leiocladum, Sorghum macrospermum, Sorghummatarankense, Sorghum miliaceum, Sorghum nigrum, Sorghum nitidum,Sorghum plumosum, Sorghum propinquum, Sorghum purpureosericeum, Sorghumstipoideum, Sorghum sudanensese, Sorghum timorense, Sorghumtrichocladum, Sorghum versicolor, Sorghum virgatum, Sorghum vulgare, orhybrids such as Sorghum x almum, Sorghum x sudangrass or Sorghum xdrummondii.

Thus, the methods and compositions can be used over a broad range ofplant species, including species from the dicot genera Brassica,Carthamus, Glycine, Gossypium, Helianthus, Jatropha, Parthenium,Populus, and Ricinus; and the monocot genera Elaeis, Festuca, Hordeum,Lolium, Oryza, Panicum, Pennisetum, Phleum, Poa, Saccharum, Secale,Sorghum, Triticosecale, Triticum, and Zea. In some embodiments, a plantis a member of the species Panicum virgatum (switchgrass), Sorghumbicolor (sorghum, sudangrass), Miscanthus giganteus (miscanthus),Saccharum sp. (energycane), Populus balsamifera (poplar), Zea mays(corn), Glycine max (soybean), Brassica napus (canola), Triticumaestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa (rice),Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris(sugarbeet), or Pennisetum glaucum (pearl millet).

In certain embodiments, the polynucleotides and vectors described hereincan be used to transform a number of monocotyledonous and dicotyledonousplants and plant cell systems, wherein such plants are hybrids ofdifferent species or varieties of a specific species (e.g., Saccharumsp. X Miscanthus sp., Sorghum sp. X Miscanthus sp., e.g., Panicumvirgatum x Panicum amarum, Panicum virgatum x Panicum amarulum, andPennisetum purpureum x Pennisetum typhoidum).

D. Transgenic Plant Phenotypes

In some embodiments, a plant expressing an aluminum tolerance-modulatingpolypeptide can have increased levels of aluminum tolerance in plants.The aluminum tolerance level can be increased by at least 2 percent,e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 45, 50, 55, 60, or more than 60 percent, as comparedto the aluminum tolerance level in a corresponding control plant thatdoes not express the transgene. As described above, aluminum tolerancecan be assessed by monitoring root growth or plant height in acidicsoils containing elevated levels of Al³⁺.

A plant in which expression of an aluminum tolerance-modulatingpolypeptide is modulated can have increased or decreased levels of seedproduction. The level can be increased or decreased by at least 2percent, e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or more than 35percent, as compared to the seed production level in a correspondingcontrol plant that does not express the transgene. Increases in seedproduction can provide improved nutritional availability in geographiclocales where intake of plant foods is often insufficient, or forbiofuel production.

Typically, a difference in the amount of aluminum tolerance (e.g., asmeasured by root growth or plant height) in a transgenic plant relativeto a control plant is considered statistically significant at p≤0.05with an appropriate parametric or non-parametric statistic, e.g.,Chi-square test, Student's t-test, Mann-Whitney test, or F-test. In someembodiments, a difference in the amount of aluminum tolerance isstatistically significant at p<0.01, p<0.005, or p<0.001. Astatistically significant difference in, for example, the amount ofaluminum tolerance in a transgenic plant compared to the amount of acontrol plant indicates that the recombinant nucleic acid present in thetransgenic plant results in altered aluminum tolerance levels.

The phenotype of a transgenic plant is evaluated relative to a controlplant. A plant is said “not to express” a polypeptide when the plantexhibits less than 10%, e.g., less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1%, 0.5%, 0.1%, 0.01%, or 0.001%, of the amount of polypeptide or mRNAencoding the polypeptide exhibited by the plant of interest. Expressioncan be evaluated using methods including, for example, RT-PCR, Northernblots, 51 RNase protection, primer extensions, Western blots, proteingel electrophoresis, immunoprecipitation, enzyme-linked immunoassays,chip assays, and mass spectrometry. It should be noted that if apolypeptide is expressed under the control of a tissue-preferential orbroadly expressing promoter, expression can be evaluated in the entireplant or in a selected tissue. Similarly, if a polypeptide is expressedat a particular time, e.g., at a particular time in development or uponinduction, expression can be evaluated selectively at a desired timeperiod.

V. Modifying Endogenous Nucleic Acids Encoding AluminumTolerance-Modulating Polypeptides

This document also features plant cells and plants in which anendogenous aluminum tolerance-modulating nucleic acid described hereinhas been modified (e.g., a regulatory region, intron, or coding regionof the aluminum tolerance-modulating nucleic acid has been modified).The aluminum tolerance of such plants is altered relative to thecorresponding level of a control plant in which the endogenous nucleicacid is not modified. Such plants are referred to herein as modifiedplants and may be used to produce, for example, increased amounts ofaluminum tolerance.

Endogenous nucleic acid can be modified by homologous recombinationtechniques. For example, sequence specific endonucleases (e.g., zincfinger nucleases (ZFNs)) and meganucleases can be used to stimulatehomologous recombination at endogenous plant genes. See, e.g., Townsendet al., Nature 459:442-445 (2009); Tovkach et al., Plant J., 57:747-757(2009); and Lloyd et al., Proc. Natl. Acad. Sci. USA, 102:2232-2237(2005). In particular, ZFNs engineered to create DNA double strandbreaks at specific loci can be used to make targeted sequence changes inendogenous plant genes. For example, an endogenous plant gene can bereplaced with a variant containing one or more mutations (e.g., producedusing site-directed mutagenesis or directed evolution). In someembodiments, site directed mutagenesis is achieved via non-homologousend joining such that after breaking DNA, endogenous DNA repairmechanisms ligate the break, often introducing slight deletions oradditions that can be screened at the cell or plant level for desiredphenotypes. Moore and Haber, Mol Cell Biol., 16(5):2164-73 (1996).

In some embodiments, endogenous nucleic acids can be modified bymethylation or demethylation such that the expression of the modifiedendogenous nucleic acid is altered. For example, a double stranded RNAcan be used to activate gene expression by targeting noncodingregulatory regions in gene promoters. See Shibuya et al., Proc Natl AcadSci USA, 106(5): 1660-1665 (2009); and Li et al., Proc Natl Acad SciUSA, 103(46):17337-42 (2006). In some embodiments, ZFNs engineered tocreate DNA double strand breaks at specific loci can be used to insert aDNA fragment having at least one region that overlaps with theendogenous DNA to facilitate homologous recombination, such that thenon-overlapping portion of the DNA fragment is integrated at the breaksite. For example, a fragment can be inserted into an endogenouspromoter and/or regulatory region at a specific site where a ZFN createsa double stranded break to alter the expression of an endogenous gene.For example, a fragment that is inserted into an endogenous gene codingregion at a specific site where a ZFN creates a double stranded breakcan result in expression of a chimeric gene. For example, a fragmentthat functions as a regulator region or promoter that is inserted intoan endogenous DNA region immediately upstream of a gene coding sequenceat a specific site where a ZFN creates a double stranded break canresult in altered expression of the endogenous gene.

In some embodiments, endogenous nucleic acids can be modified usingactivation tagging. For example, a vector containing multiple copies ofan enhancer element from the constitutively active promoter of thecauliflower mosaic virus (CaMV) ³⁵S gene can be used to activate anendogenous gene. See, Weigel et al., Plant Physiology, 122:1003-1013(2000).

In some embodiments, endogenous nucleic acids can be modified byintroducing an engineered transcription activation/repression factor(e.g., zinc finger protein transcription factor, or ZFP TF. See, forexample, the world wide web atsangamo.com/tech/techplat_over.html#whatarezfp). For example, asynthetic transcription facto sequence of a zinc finger DNA bindingdomain and a VP16 activation domain can be designed to bind to aspecific endogenous DNA site and alter expression of an endogenous gene.An engineered transcription activation/repression factor (such as ZFPTF) can activate, repress, or switch the target endogenous aluminumtolerance gene expression by binding specifically to the promoter regionor coding region of the endogenous gene. Engineered nucleases thatcleave specific DNA sequences in vivo can also be valuable reagents fortargeted mutagenesis. One such class of sequence-specific nucleases canbe created by fusing transcription activator-like effectors (TALEs) tothe catalytic domain of the FokI endonuclease. Both native and customTALE-nuclease fusions direct DNA double-strand breaks to specific,targeted sites. Christian et al., Genetics 186: 757-761 (2010).

In some embodiments, endogenous nucleic acids can be modified bymutagenesis. Genetic mutations can be introduced within regenerableplant tissue using one or more mutagenic agents. Suitable mutagenicagents include, for example, ethyl methane sulfonate (EMS),N-nitroso-N-ethylurea (ENU), methyl N-nitrosoguanidine (MNNG), ethidiumbromide, diepoxybutane, ionizing radiation, x-rays, UV rays and othermutagens known in the art. Suitable types of mutations include, forexample, insertions or deletions of nucleotides, and transitions ortransversions in the endogenous nucleic acid sequence. In oneembodiment, TILLING (Targeted Induced Local Lesions In Genomes) can beused to produce plants having a modified endogenous nucleic acid.TILLING combines high-density mutagenesis with high-throughput screeningmethods. See, for example, McCallum et al., Nat Biotechnol 18: 455-457(2000); reviewed by Stemple, Nat Rev Genet. 5(2):145-50 (2004).

In some embodiments, an endogenous nucleic acid can be modified via agene silencing technique. See, for example, the section herein regarding“Inhibition of Expression of an aluminum tolerance-ModulatingPolypeptide.”

A population of plants can be screened and/or selected for those membersof the population that have a modified nucleic acid. A population ofplants also can be screened and/or selected for those members of thepopulation that have a trait or phenotype conferred by expression of themodified nucleic acid. As an alternative, a population of plants can bescreened for those plants having a desired trait, such as a modulatedlevel of aluminum tolerance. For example, a population of progeny can bescreened for those plants having a desired level of expression of analuminum tolerance-modulating polypeptide or nucleic acid. Physical andbiochemical methods can be used to identify modified nucleic acidsand/or expression levels as described with transgenic plants. Selectionand/or screening can be carried out over one or more generations, and/orin more than one geographic location. In some cases, plants can be grownand selected under conditions which induce a desired phenotype or areotherwise necessary to produce a desired phenotype in a modified plant.In addition, selection and/or screening can be applied during aparticular developmental stage in which the phenotype is expected to beexhibited by the plant. Selection and/or screening can be carried out tochoose those modified plants having a statistically significantdifference in an aluminum tolerance level relative to a control plant inwhich the nucleic acid has not been modified. Selected or screenedmodified plants have an altered phenotype as compared to a correspondingcontrol plant, as described in the “Transgenic Plant Phenotypes” sectionherein.

Although a plant or plant cell in which an endogenous aluminumtolerance-modulating nucleic acid has been modified is not transgenicfor that particular nucleic acid, it will be appreciated that such aplant or cell may contain transgenes. For example, a modified plant cancontain a transgene for other traits, such as herbicide tolerance orinsect resistance. As another example, a modified plant can contain oneor more transgenes that, in conjuction with modifications of one or moreendogenous nucleic acids, exhibits an increase in aluminum tolerance.

As with transgenic plant cells, modified plant cells can constitute partor all of a whole plant. Such plants can be grown in the same manner asdescribed for transgenic plants and can be bred or propagated in thesame manner as described for transgenic plants.

VI. Plant Breeding

Genetic polymorphisms that are useful in such methods include simplesequence repeats (SSRs, or microsatellites), rapid amplification ofpolymorphic DNA (RAPDs), single nucleotide polymorphisms (SNPs),amplified fragment length polymorphisms (AFLPs) and restriction fragmentlength polymorphisms (RFLPs). SSR polymorphisms can be identified, forexample, by making sequence specific probes and amplifying template DNAfrom individuals in the population of interest by PCR. For example, PCRtechniques can be used to enzymatically amplify a genetic markerassociated with a nucleotide sequence conferring a specific trait (e.g.,nucleotide sequences described herein). PCR can be used to amplifyspecific sequences from DNA as well as RNA, including sequences fromtotal genomic DNA or total cellular RNA. When using RNA as a source oftemplate, reverse transcriptase can be used to synthesize complementaryDNA (cDNA) strands. Various PCR methods are described, for example, inPCR Primer: A Laboratory Manual, Dieffenbach and Dveksler, eds., ColdSpring Harbor Laboratory Press, 1995.

Generally, sequence information from polynucleotides flanking the regionof interest or beyond is employed to design oligonucleotide primers thatare identical or similar in sequence to opposite strands of the templateto be amplified. Primers are typically 14 to 40 nucleotides in length,but can range from 10 nucleotides to hundreds of nucleotides in length.Template and amplified DNA is repeatedly denatured at a high temperatureto separate the double strand, then cooled to allow annealing of primersand the extension of nucleotide sequences through the microsatellite,resulting in sufficient DNA for detection of PCR products. If the probesflank an SSR in the population, PCR products of different sizes will beproduced. See, e.g., U.S. Pat. No. 5,766,847.

PCR products can be qualitative or quantitatively analyzed using severaltechniques. For example, PCR products can be stained with a fluorescentmolecule (e.g., PicoGreen® or OliGreen®) and detected in solution usingspectrophotometry or capillary electrophoresis. In some cases, PCRproducts can be separated in a gel matrix (e.g., agarose orpolyacrylamide) by electrophoresis, and size-fractionated bandscomprising PCR products can be visualized using nucleic acid stains.Suitable stains can fluoresce under UV light (e.g., Ethidium bromide, GRSafe, SYBR® Green, or SYBR® Gold). The results can be visualized viatransillumination or epi-illumination, and an image of the fluorescentpattern can be acquired using a camera or scanner, for example. Theimage can be processed and analyzed using specialized software (e.g.,ImageJ) to measure and compare the intensity of a band of interestagainst a standard loaded on the same gel.

Alternatively, SSR polymorphisms can be identified by using PCRproduct(s) as a probe against Southern blots from different individualsin the population. See, U. H. Refseth et al., (1997) Electrophoresis 18:1519. Briefly, PCR products are separated by length through gelelectrophoresis and transferred to a membrane. SSR-specific DNA probes,such as oligonucleotides labeled with radioactive, fluorescent, orchromogenic molecules, are applied to the membrane and hybridize tobound PCR products with a complementary nucleotide sequence. The patternof hybridization can be visualized by autoradiography or by developmentof color on the membrane, for example.

In some cases, PCR products can be quantified using a real-timethermocycler detection system. For example, Quantitative real-time PCRcan use a fluorescent dye that forms a DNA-dye-complex (e.g., SYBR®Green), or a fluorophore-containing DNA probe, such as single-strandedoligonucleotides covalently bound to a fluorescent reporter orfluorophore (e.g. 6-carboxyfluorescein or tetrachlorofluorescin) andquencher (e.g., tetramethylrhodamine or dihydrocyclopyrroloindoletripeptide minor groove binder). The fluorescent signal allows detectionof the amplified product in real time, thereby indicating the presenceof a sequence of interest, and allowing quantification of the copynumber of a sequence of interest in cellular DNA or expression level ofa sequence of interest from cellular mRNA.

The identification of RFLPs is discussed, for example, in Alonso-Blancoet al. (Methods in Molecular Biology, vol. 82, “Arabidopsis Protocols”,pp. 137-146, J. M. Martinez-Zapater and J. Salinas, eds., c. 1998 byHumana Press, Totowa, N.J.); Burr (“Mapping Genes with RecombinantInbreds”, pp. 249-254, in Freeling, M. and V. Walbot (Ed.), The MaizeHandbook, c. 1994 by Springer-Verlag New York, Inc.: New York, N.Y.,USA; Berlin Germany; Burr et al. Genetics (1998) 118: 519; and Gardiner,J. et al., (1993) Genetics 134: 917). For example, to produce a RFLPlibrary enriched with single- or low-copy expressed sequences, total DNAcan be digested with a methylation-sensitive enzyme (e.g., PstI). Thedigested DNA can be separated by size on a preparative gel.Polynucleotide fragments (500 to 2000 bp) can be excised, eluted andcloned into a plasmid vector (e.g., pUC18). Southern blots of plasmiddigests can be probed with total sheared DNA to select clones thathybridize to single- and low-copy sequences. Additional restrictionendonucleases can be tested to increase the number of polymorphismsdetected.

The identification of AFLPs is discussed, for example, in EP 0 534 858and U.S. Pat. No. 5,878,215. In general, total cellular DNA is digestedwith one or more restriction enzymes. Restriction halfsite-specificadapters are ligated to all restriction fragments and the fragments areselectively amplified with two PCR primers that have correspondingadaptor and restriction site specific sequences. The PCR products can bevisualized after size-fractionation, as described above.

In some embodiments, the methods are directed to breeding a plant line.Such methods use genetic polymorphisms identified as described above ina marker assisted breeding program to facilitate the development oflines that have a desired alteration in the aluminum tolerance trait.Once a suitable genetic polymorphism is identified as being associatedwith variation for the trait, one or more individual plants areidentified that possess the polymorphic allele correlated with thedesired variation. Those plants are then used in a breeding program tocombine the polymorphic allele with a plurality of other alleles atother loci that are correlated with the desired variation. Techniquessuitable for use in a plant breeding program are known in the art andinclude, without limitation, backcrossing, mass selection, pedigreebreeding, bulk selection, crossing to another population and recurrentselection. These techniques can be used alone or in combination with oneor more other techniques in a breeding program. Thus, each identifiedplants is selfed or crossed a different plant to produce seed which isthen germinated to form progeny plants. At least one such progeny plantis then selfed or crossed with a different plant to form a subsequentprogeny generation. The breeding program can repeat the steps of selfingor outcrossing for an additional 0 to 5 generations as appropriate inorder to achieve the desired uniformity and stability in the resultingplant line, which retains the polymorphic allele. In most breedingprograms, analysis for the particular polymorphic allele will be carriedout in each generation, although analysis can be carried out inalternate generations if desired.

In some cases, selection for other useful traits is also carried out,e.g., selection for fungal resistance or bacterial resistance. Selectionfor such other traits can be carried out before, during or afteridentification of individual plants that possess the desired polymorphicallele.

VII. Articles of Manufacture

Transgenic plants provided herein have various uses in the agriculturaland energy production industries. For example, transgenic plantsdescribed herein can be used to make animal feed and food products. Suchplants, however, are often particularly useful as a feedstock for energyproduction.

Transgenic plants described herein produce higher yields of grain and/orbiomass per hectare, relative to control plants that lack the exogenousnucleic acid or lack the modified endogenous nucleic acid when grown onsoils with a pH less than 5 and elevated aluminum levels. For example,transgenic plants described herein can have a grain yield that isincreased about 5% to about 20% (e.g., increased 5% to 10%, 5% to 15%,10% to 15%, 10% to 20%, or 15% to 20%) relative to that of controlplants lacking the exogenous nucleic acid or lacking the modifiedendogenous nucleic acid. In some embodiments, such transgenic plantsprovide equivalent or even increased yields of grain and/or biomass perhectare relative to control plants when grown under conditions ofreduced inputs such as fertilizer and/or water. Thus, such transgenicplants can be used to provide yield stability at a lower input costand/or under environmentally stressful conditions such as low pH andelevated aluminum levels.

In some embodiments, plants described herein have a composition thatpermits more efficient processing into free sugars, and subsequentlyethanol, for energy production. In some embodiments, such plants providehigher yields of ethanol, butanol, dimethyl ether, other biofuelmolecules, and/or sugar-derived co-products per kilogram of plantmaterial, relative to control plants. Such processing efficiencies arebelieved to be derived from the composition of the plant material,including, but not limited to, content of glucan, cellulose,hemicellulose, and lignin. By providing higher yields at an equivalentor even decreased cost of production, the transgenic plants describedherein improve profitability for farmers and processors as well asdecrease costs to consumers.

Seeds from transgenic plants described herein can be conditioned andbagged in packaging material by means known in the art to form anarticle of manufacture. Packaging material such as paper and cloth arewell known in the art. A package of seed can have a label, e.g., a tagor label secured to the packaging material, a label printed on thepackaging material, or a label inserted within the package, thatdescribes the nature of the seeds therein.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

VIII. Examples Example 1 Materials and Methods

Plant agar culture media lacking phosphate were prepared in 2 L Pyrexglass bottles and contained the following per 1 L: 0.78 g/L of ½×MSwithout KH₂PO₄ (Sigma Chemical Co., St. Louis, Mo.), 0.5 g/L MES (SigmaChemical Co.) and 7 g/L of 0.7% Phytagar (EM Science, Gibbstown, N.J.).Media were stirred for 30 minutes after the addition of ½ MS. The pHafter the MS was completely dissolved was close to pH 5.7, and adjustedusing KOH. The final pH of each batch was measured due to pH variabilitybetween batches. Media preparations were autoclaved at about 121° C. for25 minutes on a liquid setting. Approximately 40 mL of media were addedto each square plate (100×100×15 mm) in a laminar flow hood using aliquid dispensing device or 50 mL sterile pipette. The plates wereimmediately covered with lids to avoid contamination. Plates wereallowed to cool in a laminar flow hood for at least 1 hour, but not morethan 3 hours, with a blower on. Plates were stored in 4° C. in bags.

Liquid media lacking phosphate for use in Al assays were prepared in 2 LPyrex glass bottles and contained per 1 L: 0.78 g/L of MS salt withoutKH₂PO₄ (Sigma Chemical Co.), 0.5 g/L MES (Sigma Chemical Co.), and 6 mLof 0.1M AlCl₃ (Sigma Chemical Co.) (600 μM AlCl₃ and predicted Al⁺³activity of 160 μM). Media were stirred for 30 minutes while the pH wasadjusted slowly to pH 4.0 using 1N HCL. AlCl₃ was added afterautoclaving. The preparations were autoclaved at about 121° C. for 25minutes on a liquid setting.

Agar buffer media lacking phosphate, for use in Al assays, were preparedin 2 L Pyrex glass bottles and contained per 1 L: 0.78 g/L of MS saltwithout KH₂PO₄ (Sigma Chemical Co.), 0.5 g/L MES (Sigma Chemical Co.),and 4 g/L Phytagar (EM Science), with 1N HCL used to adjust to a pH of4.8. The media were stirred for 30 minutes after the addition Phytagarand 1 N HCL. The final pH of each batch was recorded due to pHvariability between batches. The media preparations were autoclaved atabout 121° C. for 25 minutes on liquid setting. Approximately 20 mL ofwarm agar containing Al⁺³ media were aliquoted to each of 10 horizontalwells within each hydroponic box (10 horizontal wells per box/10 seedsper horizontal well) using a 50 mL sterile pipette and allowed tosolidify for about 2 hours before layering 10 mL of cooled liquid Almedia on top over the agar media described above. A thin layer of theliquid media floated on and was suspended over the agar solidified medialacking phosphate and containing Al⁺³. The hydroponic boxes wereimmediately covered with lids to avoid contamination and evaporation ofthe liquid layer before Al⁺³ infiltrations into the solid agar layer.The hydroponic boxes were allowed to rest in the laminar flow hood forat least 1 hour, but not more than 3 hours, with a blower on (to avoidevaporation of the liquid layer during infiltration). The hydroponicboxes were stored in 4° C.

To sterilize seeds, the appropriate number of seeds from each candidateplant or ME line event were transferred to individual 2 mL tubes and 1mL 30% Clorox bleach was added to each tube and agitated manually for atleast 5 minutes until all seeds become suspended. Bleach solution thenwas discarded and seeds were rinsed aseptically with sterile nanopurewater at least 4 times or until the seeds were thoroughly rinsed.

Example 2 Al⁺³ Assay for Transgenic Arabidopsis thaliana

Forty-eight T₃ generation seeds of each of transgenic ME02064 events-01,-04, -03, plus an empty vector control (SR00559), were sterilized andplated on agar media lacking phosphate hydroponically suspended overAl⁺³ liquid media (see Example 1). In each mini-hydroponic box, 48control seeds plus 48 ME02064 seeds were sown. Boxes were kept in a 4°C. refrigerator in the dark for 3 days to promote uniform germination.After 3 days of cold treatment, the boxes were placed horizontally in aConviron growth chamber as described above. The plates were scannedusing a fluorescence imager scanner (Technologica Ltd, Colchester, UK)at 7 days and 14 days after sowing seeds. Plants were scored for growthwhen Al toxicity (e.g., stunted root growth and reduced rosette area)became apparent, usually around 10 days.

The seedling area and fluorescence intensity (Fv/Fm) were quantified ina pooled manner. Fv/Fm measurements as well as rosette area growth foreach individual plant were not taken. Rather, the -01, -04, & -03seedling populations (48 seedlings) were compared to the pooled control(48 seedlings) within the same mini-hydroponic box. Total rosette growthwas measured for 48 seedlings from transgenic lines and for 48 seedlingsfrom control lines. Stress that resulted in decreases in the quantumefficiency of photochemistry (open PSII reaction centers), determined byFv/Fm of dark adapted plants, was considered an indicator of inhibitionof photosynthesis by Al Basta resistance was not tested as other assayshad indicated that these lines were transgenic.

Rosette area was larger at 10 days in all transgenic events of -01, -04,& -03 when compared to empty vector controls (SR00559) as shown inTable 1. However, no visual difference in root growth was observed (datanot shown). Roots for both transgenics and controls were all severelystunted, failing to penetrate from the agar media support into the highAl⁺³ liquid media. The mean Fv/Fm appeared to be moderately higher intransgenic events-01, -04, & -03 relative to controls, but there was nostatistically significant difference.

TABLE 1 60 μM AlCl₃ 10 days ME02064-01T3 SR00559 Sum of Rosette 587.74478.23 Areas (mm²) Mean Fv/Fm 0.722 0.713 60 μM AlCl₃ 10 daysME02064-04T3 SR00559 Sum of Rosette 676.19 619.3 Areas (mm²) Mean Fv/Fm0.775 0.763 60 μM AlCl₃ 10 days ME02064-03T3 SR00559 Sum of Rosette578.23 436.96 Areas (mm²) Mean Fv/Fm 0.712 0.717

Example 3 Al⁺³ assay for Transgenic Switchgrass Plants

A T-DNA binary vector containing Ceres Clone 375578 (SEQ ID NO:352) wasintroduced into switchgrass by Agrobacterium-mediated transformationessentially as described in Richards et al., Plant Cell. Rep. 20:48-54(2001) and Somleva, et al., Crop Sci. 42:2080-2087 (2002). The presenceof the transgene was confirmed by PCR. Plants comprising the transgenewere grown in acidified soil containing high levels of Al⁺³. The soilwas made by dissolving AlCl₃ in water and watering the soil with thealuminized water. The soil then was acidified using pH 4 buffered water,to release the toxic aluminum ion. Because the acidity of the soildetermines how much of the Al⁺³ is present as the toxic free ion, the Alvalue was variable. Switchgrass control non-transgenic plants (Wt) andtransgenic (CeresClone 375578) plants were placed in soil in separatepots in a growth room and allowed to grow under standard growth roomconditions. Non-transgenic plants were generated from calli withoutco-cultivation with agrobacteria. There was one plant per pot. Theplantlets were watered with one of the following three treatments:Treatment 1: 2 L of water, pH 7.26; Treatment 2: 2 L of water, pH 4.0;and Treatment 3: 2 L of water, pH 4.0+˜11 g/Kg soil Al⁺³(˜621 μM). Thesize of the transgenics and controls appeared similar when the treatmentwas started but no measurements were taken. After 16 days, seedlingswere harvested, washed, and then dried in a drying oven. The dry weightbiomass was measured for each plant.

Transgenic aerial plant growth was greater in the transgenic switchgrassevents than in the controls as indicated by an increase in whole plantweight (shoots and roots). The increase was statistically significant ata P value of 0.05 when compared to wild-type plants (see FIG. 5, Tables2 and 3). Root growth in the transgenic lines was visually more robustthan wild-type controls. These results indicate that switchgrasscontaining Ceres Clone 375578 can survive in acidified soil having analuminum chloride concentration of about 11 g/kg soil.

TABLE 2 Whole Plant Weight (Shoots + Roots) Treatment 1 Treatment 2Treatment 3 Wt 375578 Wt 375578 Wt 375578 Mean Weight 1.23 1.4 1.33 1.390.73 1.44 (g) Standard Error 0.23 0.17 0.07 0.07 0.23 0.13 Median 1 1.31.2 1.3 0.8 1.25 Standard 0.40 0.55 0.12 0.23 0.40 0.42 Deviation Sample0.16 0.3 0.01 0.05 0.16 0.18 Variance Skewness 1.73 0.30 −1.73 0.35 0.721.04 Range 0.7 1.4 0.2 0.6 0.8 1.3 Minimum 1 0.8 1 1.1 0.3 1 Maximum 1.72.2 1.2 1.7 1.1 2.3 Sum 3.7 14 3.4 13.9 2.2 14.4 Count 3 10 3 10 3 10

TABLE 3 t-Test: Two-Sample Assuming Unequal Variances of Plants underTreatment 3 Wild type Control, pH 4 375578 Mean Whole Plant Weight(Shoots + Roots) (g) 0.73 1.44 Variance 0.16 0.18 Observations 3 10Hypothesized Mean Difference 0 Df 3 t Stat −2.63 P (T <= t) one-tail0.04 t Critical one-tail 2.35 P (T <= t) two-tail 0.08 t Criticaltwo-tail 3.18

The results also indicate that wild type plants watered with Treatment 3(pH 4 but without AlCl₃) had a whole plant weight that was less than thewhole plant dry weight of wild type plants watered with Treatment 3. Theresults also indicate that the decrease in whole plant dry weight seenunder Treatment 3 conditions was a result of Al⁺³ toxicity, becausewild-type plants grown under Treatment 2 conditions (pH 4 but withoutAlCl₃) showed no such decrease in whole plant dry weight (see Table 3).

Example 4 Germination of Rice Seedlings in Media Containing Aluminum

In this example, the role of CeresClone 24255 (Os713, 3 events),CeresClone 1752915 (Os825, 3 events), and CeresClone 11684 (Os879, 3events) was assessed in rice seeds germinated in the presence ofaluminum. Seeds of wild-type internal control (null segregant) andhomozygous transgenic plants were placed on two separate plates for eachevent. Each plate had two rows of 12 seeds aligned in parallel. Externalwild-type seed were sown at the same time under control (no aluminumchloride) conditions using the same format for comparison of rate ofgermination and growth to that of Al⁺³ conditions.

Unsterilized, de-husked seeds were germinated under normal oraluminum-stress conditions as follows. Normal conditions included aplate (150 mm×100 mm×15 mm) containing 30 mL of ½ MS medium withoutphosphate and nitrogen, no sugar, 4 g agar/L pH 5.7. Aluminum-stressconditions included a plate (150 mm×100 mm×15 mm) containing 20 mL of ½MS medium without phosphate and nitrogen, no sugar, 4 g agar/L pH 5.7,infiltrated with 10 mL AlCl₃ liquid infiltration medium containing 600μM AlCl₃ (which corresponds to a predicted ion activity of 160 μM onplates) in ½ MS pH 4. The AlCl₃ liquid infiltration medium was added ontop of the aluminum medium and allowed to settle for 24 hours beforeseed placement on the surface. AlCl₃ liquid infiltration mediumconsisted of: ½ MS media without phosphate or nitrogen; no sugar oragar; and 600 μM AlCl₃, with the pH adjusted to 4.0 with 1N HCl.Predicted Al⁺³ activities were confirmed with GEOCHEM-EZ, amulti-purpose chemical speciation program. See, Shaff et al., Plant Soil330: 207-214 (2010).

Plates with lids were not sealed but were placed inside large ziplockbags to maintain humidity in growth chamber (25° C., 70% humidity for 8hours light/16 hours dark). At 13 days, seedlings were measured forplant height and root length. See Tables 4 and 5. For Os713, plantheight and root length were not significantly different between thetransgenic plants and non-transgenic controls. For Os825 event 9, asignificant increase in plant height was observed in comparison to thepooled non-transgenic control. For Os879 event 7, a significant increasein plant height was observed in comparison to the internalnon-transgenic control. For Os879 event 4, a significant increase inplant height was observed in comparison to the internal non-transgeniccontrol as well as a significant increase in root length in comparisonto the pooled non-transgenic control. For Os870 event 8, a significantincrease in root length was observed in comparison to the internal andpooled non-transgenic controls.

TABLE 4 Plant Height after 13 d Aluminum Treatment Copy number #ofTransgenics Non-transgenics P-value of plants Standard #r of Standard #of Internal Pooled Genotype transgene tested Average Deviation plantsAverage Deviation plants NT NT OS713-07 1 24 5.11 5.01 12 6.73 4.27 120.41 0.74 OS713-05 1 24 2.92 4.47 12 1.91 2.62 12 0.50 0.22 OS713-03 124 6.42 5.63 12 5.21 3.43 12 0.53 0.23 All OS713 4.81 5.13 36 4.62 3.9736 0.86 OS825-13 1 24 3.89 5.15 12 2.09 2.55 12 0.28 0.61 OS825-10 1 243.95 4.26 12 3.15 4.43 12 0.66 0.56 OS825-09 1 24 5.95 4.95 12 4.28 4.3612 0.39 0.05 All OS825 4.60 4.76 36 3.17 3.88 36 0.23 OS879-08 1 24 5.234.34 12 2.88 3.96 12 0.18 0.10 OS879-07 1 24 4.88 4.50 12 1.28 2.81 120.03 0.18 OS879-04 1 24 5.72 4.63 12 5.03 3.73 12 0.69 0.05 All OS8795.27 4.38 36 3.07 3.77 36 0.02

TABLE 5 Root Length after 13 d Aluminum Treatment TransgenicsNon-transgenics P-value Number of Standard Number of Standard Number ofInternal Pooled Genotype plants tested Average Deviation plants AverageDeviation plants NT NT OS713-07 24 0.79 0.83 12 1.5 0.87 12 0.059 0.97OS713-05 24 0.75 1.08 12 0.2 0.36 12 0.11 0.87 OS713-03 24 1.2 1.08 120.7 0.50 12 0.16 0.18 0.92 1.00 36 0.8 0.81 36 0.59 OS825-13 24 0.380.64 12 0.28 0.47 12 0.67 0.42 OS825-10 24 0.38 0.87 12 0.73 1.12 120.41 0.46 OS825-09 24 0.58 0.51 12 0.79 0.84 12 0.47 0.96 0.45 0.68 360.60 0.86 36 0.35 OS879-08 24 0.85 0.72 12 0.29 0.45 12 0.03 0.005OS879-07 24 0.53 0.59 12 0.16 0.37 12 0.08 0.23 OS879-04 24 0.94 0.78 120.57 0.42 12 0.16 0.002 0.78 0.71 36 0.34 0.44 36 0.002

Example 5 Seedling Growth of Transgenic Rice Containing CeresClone375578, CeresClone 24255, and CeresClone 11684 in Rice

The experiments described in Example 4 were repeated using transgenicrice events for CeresClone 375578 (Line 745282, one of 7 events),CeresClone 24255 (Os713, 3 events), and CeresClone 11684 (Os879, 3events). After germination under normal or aluminum conditions asdescribed in Example 4, and incubation for 10 or 11 days in the growthchamber, surviving seedlings were transplanted to soil (60% SunshineProfessional Mix (with vermiculite); 40% Turface; 1 tbs/3 L Osmocote;1.5 tbs/3 L of Bone Meal; and 0.5 tbs/3 L Marathon).

Plants were genotyped as follows. Plants were allowed to recover untilthe two leaf stage then genotyped by cutting 5 to 6 mm leaf segmentsfrom the largest of the two leaves and placed onto Kanamycin mediumplates containing 160 mg/L of Kanamycin (¼ MS basal salt medium; 4.5 g/Lof Phytoagar; 100 μL/L of Tween 80; and adjusted to pH 5.7 with 1N KOH.Medium was sterilized under the wet cycle for 30 minutes at 120° C. andallowed to cool before adding Kanamcyin to a final concentration of 160mg/L). Plates were sealed in Ziplock bags and placed in a growth chamberat about 26±2° C. under normal growing light regime (16 h light and 8 hdark).

Plates were removed from the chamber during a light period and scannedstarting at 4d of treatment. If unclear, the plates were re-scanned at 5or 6 days post treatment. Plates were scanned for photosyntheticefficiency (PE) quantification to determine genotype. Generally, PE is aparameter (Fv/Fm, the ratio of variable florescence over the maximumflorescence value) used to indicate the quantum efficiency of thephotosystem type II (PSII) reactions within the plants chloroplasts. TheFv/Fm parameter is an indication of photosynthetic tissue health.Healthy tissue samples typically achieve an Fv/Fm value of approximately0.7-0.85. Lower values are observed if a sample is exposed to a bioticor abiotic stress factor that reduce the capacity for photochemicalquenching of energy within PSII.

Images of chlorophyll fluorescence and the florescence parameters wereobtained with Chlorophyll Fluorescence imager (Technologica, UK) usingthe manufacturer's instructions for light-adapted materials.Representative regions of the image, e.g., distant from the any cut endto avoid damage effects, were chosen for reading the Fv/Fm values. PCRanalysis was used to confirm the presence of transgenes.

After genotyping, plants were grown in soil and treated with eitherwater (Normal) or with acidified water (˜pH 4.0) containing 600 μMAlCl₃. After 19 days, root length and plant height were measured. Rootand plant height data for transgenic plants were statistically comparedto non-transgenic (NT) internal segregant lines, pooled NT internalsegregant lines, external NT lines, and all NT lines. Comparisons werealso made to plants not treated with AL containing water.

Tables 6 and 7 contain the results of line 745282 (7 events) for rootexpansion at 19 days and plant height at 19 days, respectively. For line745282, a significant increase in root expansion was observed for events745282, 745284, 745252, and 745307; a significant increase in plantheight was observed for events 745284 and 745236.

Tables 8 and 9 contain the results for Os713 and Os879 for rootexpansion at 19 days and plant height at 19 days, respectively. ForOs879, a significant increase in plant height was observed for event 7.

TABLE 6 Root expansion (cm) under Aluminum treatment 19 d TransgenicsNon-transgenics P-value Number Number Line Pooled Standard of Standardof Internals Internals External Genotype Average Deviation plantsAverage Deviation plants NT NT NT Internal + External 745282 4.55 2.9 114.2 2.68 5 0.78 0.41 0.006 0.10 745284 6.38 2.05 8 4.06 1.82 8 0.03 0.013.83E−05 0.001 745236 2.23 3.14 13 4.17 3.82 3 0.39 0.09 0.90 0.226745252 5 2.96 7 3.06 3.26 9 0.24 0.30 0.011 0.083 745306 5.6 3.44 5 6.292.1 11 0.62 0.18 0.008 0.050 745312 7.02 2.16 16 n/a n/a n/a n/a9.93E−05 3.58E−08 4.25E−07 745307 5.19 2.9 8 4.13 2.4 8 0.44 0.20 0.0050.043 All transgenics 5.1 3.03 68 5.1 9.06 0.042 0.0001 0.001 Pooled NT3.833 2.59 33 External WT 2.347 2.01 23 Internal + External 3.22 2.46 56

TABLE 7 Plant height (cm) under Aluminum treatment 19 d Copy NumberTransgenics Non-transgenics P-value number of Number Number Line PooledInternal + of plants Standard of Standard of Internals InternalsExternal External Genotype transgene tested Average Deviation plantsAverage Deviation plants NT NT NT NT 745282 1 16 21.45 2.9 11 24.3 7.985 0.32 0.38 0.0002 0.293 745284 1 16 25.63 4.24 8 21.25 3.01 8 0.0320.08 3.66E−06 0.005 745236 2 16 23.23 2.01 13 22.67 3.06 3 0.693 0.647.56E−07 0.026 745252 1 16 23.86 7.78 7 21.83 3.76 9 0.502 0.56 0.00090.076 745306 1 16 23.96 3.33 5 25 5.56 11 0.707 0.51 0.0004 0.0958745312 2 16 23.98 1.92 16 n/a n/a n/a n/a 0.23 1.14E−08 0.003 745307 116 21.44 2.29 8 23.94 2.88 8 0.075 0.43 8.77E−04 0.367 All transgenics 1& 2 112 23.31 9.42 68 0.42 8.38E−11 0.0003 Pooled NT 0 33 22.67 9.53 33External WT 0 23 15.2 9.54 23 Internal + External 0 56 19.59 4.99 56

TABLE 8 Root Length after 19 d aluminum treatment TransgenicsNon-transgenics P-value Standard Number Standard Number Internal PooledExternal Genotype Average Deviation of plants Average Deviation ofplants NT NT WT Internal + External W2 (939-006) 6.1 1.55 7 4.4 1.35 30.14 0.49 0.22 713-3 5.32 0.53 6 6.13 1.31 4 0.21 0.95 0.55 0.75 713-54.63 1.60 8 4 0 2 0.61 0.36 0.19 0.16 713-7 4.19 1.93 8 5 1 0.70 0.210.11 0.06 879-4 4.33 1.22 9 7 1 0.07 0.33 0.04 0.06 879-7 5.56 1.81 9 41 0.44 0.63 0.95 0.80 879-8 4.29 0.70 7 4.8 1.31 3 0.43 0.23 0.02 0.04All T713 4.65 1.54 22 All T879 4.76 1.44 25 Pooled NT 713_879 5.24 1.3512 Pooled NT_713 5.36 1.38 7 Pooled NT_879 5.08 1.46 5 External NT WT5.6 1.02 7 All NT SAP + Externals 5.37 1.22 19 External + NT713 5.481.19 14 External + NT879 5.38 1.19 12 All T713_Untreated 7.93 2.09 7 AllT879_Untreated 7.7 1.86 8 All T W2_Untreated 5.44 1.40 8T713_879_Untreated 7.09 1.46 11 NT713_879_Untreated 7 1.91 7

TABLE 9 Plant height after 19 d aluminum treatment Copy NumberTransgenics Non-transgenics P-value number of of plants Standard NumberStandard Number Internal Pooled External Internal + Genotype transgenetested Average Deviation of plants Average Deviation of plants NT NT WTExternal W2 (939-006) 1 10 16.17 0.99 7 15.33 2.04 3 0.39 0.20 0.24713-3 1 10 18.3 1.01 6 17.4 1.37 4 0.26 0.00 0.06 0.13 713-5 1 10 16.761.60 8 17.15 0.92 2 0.76 0.55 0.11 0.35 713-7 1 10 15.06 6.78 8 16.50.135 2 0.85 0.43 0.54 0.99 879-4 1 10 14.78 2.89 9 17.9 1 0.34 0.070.45 0.93 879-7 1 10 17.94 2.06 9 16 1 0.40 0.85 0.04 0.13 879-8 1 1018.09 1.71 7 18.23 2.87 3 0.92 0.75 0.06 0.15 All T713 1 16.56 4.26 22All T879 1 16.844 2.73 25 Pooled NT 713_879 0 17.42 1.60 12 Pooled NT_713 0 17.2 1.09 7 Pooled NT_879 0 17.72 2.25 5 External NT WT 0 12.966.19 7 All NT SAP + Externals 15.77 4.38 19 External + NT713 15.08 4.8014 External + NT879 14.94 5.36 12 All T713_Untreated 18.68 1.11 7 AllT879_Untreated 16.63 1.41 8 All T W2_Untreated 17.89 1.61 8T713_879_Untreated 17.58 1.63 15 NT713_879_Untreated 18.41 2.86 7

Example 6 Determination of Functional Homologs by Reciprocal BLAST®

A candidate sequence was considered a functional homolog of a referencesequence if the candidate and reference sequences encoded proteinshaving a similar function and/or activity. A process known as ReciprocalBLAST® (Rivera et al., Proc. Natl. Acad. Sci. USA, 95:6239-6244(1998))was used to identify potential functional homolog sequences fromdatabases consisting of all available public and proprietary peptidesequences, including NR from NCBI and peptide translations from Ceresclones.

Before starting a Reciprocal BLAST® process, a specific referencepolypeptide was searched against all peptides from its source speciesusing BLAST® in order to identify polypeptides having BLAST® sequenceidentity of 80% or greater to the reference polypeptide and an alignmentlength of 85% or greater along the shorter sequence in the alignment.The reference polypeptide and any of the aforementioned identifiedpolypeptides were designated as a cluster.

The BLAST® version 2.0 program from Washington University at SaintLouis, Missouri, USA was used to determine BLAST® sequence identity andE-value. The BLAST® version 2.0 program includes the followingparameters: 1) an E-value cutoff of 1.0e-5; 2) a word size of 5; and 3)the -postsw option. The BLAST® sequence identity was calculated based onthe alignment of the first BLAST® HSP (High-scoring Segment Pairs) ofthe identified potential functional homolog sequence with a specificreference polypeptide. The number of identically matched residues in theBLAST® HSP alignment was divided by the HSP length, and then multipliedby 100 to get the BLAST® sequence identity. The HSP length typicallyincluded gaps in the alignment, but in some cases gaps were excluded.

The main Reciprocal BLAST® process consists of two rounds of BLAST®searches; forward search and reverse search. In the forward search step,a reference polypeptide sequence, “polypeptide A,” from source speciesSA was BLASTed® against all protein sequences from a species ofinterest. Top hits were determined using an E-value cutoff of 10-5and asequence identity cutoff of 35%. Among the top hits, the sequence havingthe lowest E-value was designated as the best hit, and considered apotential functional homolog or ortholog. Any other top hit that had asequence identity of 80% or greater to the best hit or to the originalreference polypeptide was considered a potential functional homolog orortholog as well. This process was repeated for all species of interest.

In the reverse search round, the top hits identified in the forwardsearch from all species were BLASTed® against all protein sequences fromthe source species SA. A top hit from the forward search that returned apolypeptide from the aforementioned cluster as its best hit was alsoconsidered as a potential functional homolog.

Functional homologs were identified by manual inspection of potentialfunctional homolog sequences. Representative functional homologs for SEQID NOs: 353, 237, 451, and 2 are shown in FIGS. 1-4, respectively.Additional exemplary homologs are correlated to certain Figures in theSequence Listing.

Example 7 Determination of Functional Homologs by Hidden Markov Models

Hidden Markov Models (HMMs) were generated by the program HMMER 2.3.2.To generate each HMM, the default HMMER 2.3.2 program parameters,configured for global alignments, were used.

An HMM was generated using the sequences shown in FIG. 1 as input. Thesesequences were fitted to the model and a representative HMM bit scorefor each sequence is shown in the Sequence Listing. Additional sequenceswere fitted to the model, and representative HMM bit scores for any suchadditional sequences are shown in the Sequence Listing. The resultsindicate that these additional sequences are functional homologs of SEQID NO: 353.

The procedure above was repeated and an HMM was generated for each groupof sequences shown in FIGS. 2-4, using the sequences shown in eachFigure as input for that HMM. A representative bit score for eachsequence is shown in the Sequence Listing. Additional sequences werefitted to certain HMMs, and representative HMM bit scores for suchadditional sequences are shown in the Sequence Listing. The resultsindicate that these additional sequences are functional homologs of thesequences used to generate that HMM.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of increasing plant yield in soilcontaining elevated levels of Al³⁺, said method comprising growing aplant comprising an exogenous nucleic acid on the soil having theelevated level of Al³⁺, said exogenous nucleic acid comprising aregulatory region operably linked to a nucleotide sequence encoding apolypeptide having 95 percent or greater sequence identity to the aminoacid sequence set forth in SEQ ID NO:2, wherein yield of said plant isincreased as compared to the corresponding yield of a control plant thatdoes not comprise said nucleic acid, wherein the regulatory region isheterologous with respect to the nucleotide sequence, and wherein theplant belongs to the Poaceae.
 2. A method of increasing plant yield insoil containing elevated levels of Al³⁺, said method comprising growinga plant comprising an exogenous nucleic acid on the soil having theelevated level of Al³⁺, said exogenous nucleic acid comprising aregulatory region operably linked to a nucleotide sequence having 95percent or greater sequence identity to the nucleotide sequence setforth in SEQ ID NO: 1, wherein yield of said plant is increased ascompared to the corresponding yield of a control plant that does notcomprise said nucleic acid, wherein the regulatory region isheterologous with respect to the nucleotide sequence, and wherein theplant belongs to the Poaceae.
 3. The method of claim 1, wherein saidnucleotide sequence encodes a polypeptide having 99 percent or greatersequence identity to said amino acid sequence set forth in SEQ ID NO:2.4. The method of claim 1, wherein said nucleotide sequence encodes apolypeptide having 98 percent or greater sequence identity to said aminoacid sequence set forth in SEQ ID NO:2.
 5. The method of claim 2,wherein said nucleotide sequence has 99 percent or greater sequenceidentity to the nucleotide sequence set forth in SEQ ID NO:1.
 6. Themethod of claim 2, wherein said nucleotide sequence has 98 percent orgreater sequence identity to the nucleotide sequence set forth in SEQ IDNO:1.
 7. The method of claim 1, wherein said nucleotide sequence encodesthe polypeptide set forth in SEQ ID NO:2.
 8. The method of claim 1, saidmethod further comprising harvesting biomass from said plant.
 9. Themethod of claim 1, wherein said regulatory region is a promoter.
 10. Themethod of claim 9, wherein said promoter is selected from the groupconsisting of YP0092, PT0676, PT0708, PT0613, PT0672, PT0678, PT0688,PT0837, the napin promoter, the Arcelin-5 promoter, the phaseolin genepromoter, the soybean trypsin inhibitor promoter, the ACP promoter, thestearoyl-ACP desaturase gene promoter, the soybean a′ subunit of/3-conglycinin promoter, the oleosin promoter, the 15 kD zein promoter,the 16 kD zein promoter, the 19 kD zein promoter, the 22 kD zeinpromoter, the 27 kD zein promoter, the Osgt-1 promoter, the beta-amylasegene promoter, the barley hordein gene promoter, p326, YP0144, YP190,p13879, YP0050, p32449, 21876, YP0158, YP0214, YP0380, PT0848, PT0633,the cauliflower mosaic virus (CaMV) 35S promoter, the mannopine synthase(MAS) promoter, the 1′ or 2′ promoters derived from T-DNA ofAgrobacterium tumefaciens, the figwort mosaic virus 34S promoter, riceactin promoter, maize ubiquitin-1 promoter, ribulose-1, 5-bisphosphatecarboxylase (RbcS) promoter, the pine cab6 promoter, the Cab-1 genepromoter from wheat, the CAB-1 promoter from spinach, the cab1R promoterfrom rice, the pyruvate orthophosphate dikinase (PPDK) promoter fromcorn, the tobacco Lhcb1*2 promoter, the Arabidopsis thaliana SUC2sucrose-H symporter promoter, and a thylakoid membrane protein promoterfrom spinach, and PT0585.
 11. The method of claim 1, wherein said plantcomprising said exogenous nucleic acid has an improved growth raterelative to a corresponding plant that does not comprise said nucleicacid.
 12. The method of claim 1, wherein said plant comprising saidexogenous nucleic acid has improved vegetative growth relative to acorresponding plant that does not comprise said nucleic acid.
 13. Amethod of increasing tolerance of a plant to elevated levels ofaluminum, said method comprising a) introducing into a plurality ofplant cells an exogenous nucleic acid comprising a regulatory regionoperably linked to a heterologous nucleic acid sequence encoding apolypeptide having 95 percent or greater sequence identity to the aminoacid sequence set forth in SEQ ID NO:2; b) producing a plant from saidplant cell; and c) growing said plant on soil having an elevated levelof Al³⁺, wherein said plant has increased yield as compared to that of acontrol plan that does not comprise said nucleic acid, and wherein theplant belongs to the Poaceae.
 14. A method of increasing tolerance of aplant to elevated levels of aluminum, said method comprising a)introducing into a plurality of plant cells an exogenous nucleic acidcomprising a regulatory region operably linked to a heterologous nucleicacid sequence encoding a polypeptide having 95 percent or greatersequence identity to the amino acid sequence set forth in SEQ ID NO:2;and b) selecting a plant produced from said plurality of plant cellsthat has an increased tolerance to elevated A1 ³⁺as compared to thetolerance in a corresponding control plant that does not comprise saidexogenous nucleic acid, wherein the plant belongs to the Poaceae. 15.The method of claim 14, wherein regulatory region is a promoter.
 16. Themethod of claim 1, wherein the plant is a rice plant.
 17. The method ofclaim 1, wherein the plant is a wheat plant.
 18. The method of claim 1,wherein the plant is a corn plant.
 19. The method of claim 1, whereinthe plant is a sorghum plant.
 20. The method of claim 1, wherein theplant is a switchgrass plant.